Analytical ultracentrifugation for characterization of recombinant viral particles

ABSTRACT

Provided herein are methods to characterize preparations of recombinant viral particles using analytical ultracentrifugation. Recombinant viral particles include recombinant adeno-associated viral particles, recombinant adenoviral particles, recombinant lentiviral particles and recombinant herpes simplex virus particles. Variant species of recombinant viral particles including empty capsids and recombinant viral particles with variant genomes (e.g., truncated genomes, aggregates, recombinants) can be identified and quantitated. The methods can be used to characterize preparations of recombinant viral particles regardless of the sequence of the recombinant viral genome or the serotype of the recombinant viral capsid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase application under 35 U.S.C. § 371of International Application No. PCT/US2016/013947 filed Jan. 19, 2016,which claims priority to U.S. Provisional Application No. 62/105,714,filed Jan. 20, 2015, each of which is incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

The present invention relates to methods to characterize recombinantviral vectors; e.g., recombinant adeno-associated viral (AAV) particles,recombinant adenoviral (rAd) particles, recombinant lentiviral particlesand recombinant Herpes simplex viral (rHSV) particles using analyticalultracentrifugation.

BACKGROUND OF THE INVENTION

Recombinant viruses show great promise and utility as a vehicle todeliver therapeutic nucleic acids for gene therapy applications. Anumber of different recombinant viruses are used in these gene therapyapplications based on a number of factors including the size of thenucleic acid to be delivered, the target cell or tissue to deliver thenucleic acid, the need for short or long term expression of thetherapeutic nucleic acid, and integration of the therapeutic nucleicacid into the recipient's genome. Examples of viruses used in genetherapy applications include adeno-associated virus (AAV), adenovirus,lentivirus and herpes simplex virus (HSV).

The generation of recombinant viral vectors for the clinic requires ananalytical method that monitors drug product quality with regard tohomogeneity, purity and consistency of manufacturing, yet to date nomethod to support such a characterization has been established.Typically, the DNA content of recombinant viral DNA viral vectors ismeasured by Southern blot analysis using a sequence specific probe.Viral capsids or envelopes may be characterized by immunoassay using anantibody that binds specifically to a capsid or envelope protein of aparticular recombinant virus. For example, Steinbach, S et al., (1997)J. Gen. Virol., 78:1453-1462 provides an immunoassay for rAAV serotypes.What is needed is a generic assay to characterize recombinant viralpreparations regardless of the nucleic acid sequence of the recombinantviral genome or the serotype of the capsid.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

In some aspects, the invention provides methods of characterizing apreparation of recombinant viral particles comprising the steps of a)subjecting the preparation to analytical ultracentrifugation underboundary sedimentation velocity conditions wherein the sedimentation ofrecombinant viral particles is monitored at time intervals, b) plottingthe differential sedimentation coefficient distribution value (C(s))versus the sedimentation coefficient in Svedberg units (S), and c)integrating the area under each peak in the C(s) distribution todetermine the relative concentration of each peak, wherein each peakrepresents a species of recombinant viral recombinant viral particle.

In some aspects, the invention provides methods to assess vector genomeintegrity of recombinant viral particles in a preparation of recombinantviral particles comprising a) subjecting the preparation to analyticalultracentrifugation under boundary sedimentation velocity conditionswherein the sedimentation of recombinant viral particles is monitored attime intervals, b) plotting the differential sedimentation coefficientdistribution value C(s) versus the sedimentation coefficient in Svedbergunits (S), and c) identifying species of recombinant viral particles inthe preparation by presence of peaks on the plot corresponding to an Svalue, wherein the genome size of a particular species of recombinantviral recombinant viral particles is calculated by comparing the S valueof the species to a standard curve generated by S values of recombinantviral particles comprising encapsidated viral genomes of knownnucleotide sizes. In some embodiments, the methods further compriseintegrating the area under each peak in the C(S) distribution todetermine the relative concentration of each species of recombinantviral recombinant viral particles.

In some aspects, the invention provides methods to determine thepresence of empty capsids or capsid particles comprising variant sizedrecombinant viral genomes in a preparation of recombinant viralparticles comprising the steps of a) subjecting the preparation toanalytical ultracentrifugation under boundary sedimentation velocityconditions wherein the sedimentation of recombinant viral particles ismonitored at time intervals, and b) plotting the differentialsedimentation coefficient distribution value (C(s)) versus thesedimentation coefficient in Svedberg units (S), wherein the presence ofone or more peaks other than the peak for full capsid particlescomprising intact recombinant viral genomes indicates that presence ofcapsid particles comprising variant sized genomes and/or empty capsids.

In some aspects, the invention provides, methods of measuring therelative amount empty capsids in a preparation of recombinant viralparticles comprising the steps of a) subjecting the preparation toanalytical ultracentrifugation under boundary sedimentation velocityconditions wherein the sedimentation of recombinant viral particles ismonitored at time intervals, b) plotting the differential sedimentationcoefficient distribution value (C(s)) versus the sedimentationcoefficient in Svedberg units (S), c) integrating the area under eachpeak in the C(S) distribution to determine the relative concentration ofeach species of recombinant viral particles, and d) comparing the amountof recombinant viral particles having an S value corresponding to emptycapsid particles to the amount of recombinant viral particles having anS value corresponding to recombinant viral particles comprising intactviral genomes or the total amount of recombinant viral particles in thepreparation.

In some aspects, the invention provides methods of measuring therelative amount of capsid particles comprising variant recombinant viralgenomes or empty viral capsid particles in a preparation of recombinantviral particles comprising the steps of a) subjecting the preparation toanalytical ultracentrifugation under boundary sedimentation velocityconditions wherein the sedimentation of recombinant viral particles ismonitored at time intervals, b) plotting the differential sedimentationcoefficient distribution value (C(s)) versus the sedimentationcoefficient in Svedberg units (S), c) integrating the area under eachpeak in the C(S) distribution to determine the relative concentration ofeach species of recombinant viral particles, d) comparing the amount ofrecombinant viral particles having an S values that do not correspond torecombinant viral particles comprising intact viral genomes to theamount of recombinant viral particles having an S value that correspondsto recombinant viral particles comprising intact viral genomes or to thetotal amount of recombinant viral particles in the preparation.

In some aspects, the invention provides methods of measuring therelative amount of capsid particles comprising variant recombinant viralgenomes in a preparation of recombinant viral particles comprising thesteps of a) subjecting the preparation to analytical ultracentrifugationunder boundary sedimentation velocity conditions wherein thesedimentation of recombinant viral particles is monitored at timeintervals, b) plotting the differential sedimentation coefficientdistribution value (C(s)) versus the sedimentation coefficient inSvedberg units (S), c) integrating the area under each peak in the C(S)distribution to determine the relative concentration of each species ofrecombinant viral particles, d) comparing the amount of recombinantviral particles having an S values that do not correspond to recombinantviral particles comprising intact viral genomes or empty capsidparticles to the total amount of recombinant viral particles in thepreparation.

In some aspects, the invention provides methods of measuring therelative amount of recombinant viral particles comprising intact viralgenomes in a preparation of recombinant viral particles comprising thesteps of a) subjecting the preparation to analytical ultracentrifugationunder boundary sedimentation velocity conditions wherein thesedimentation of recombinant viral particles is monitored at timeintervals, b) plotting the differential sedimentation coefficientdistribution value (C(s)) versus the sedimentation coefficient inSvedberg units (S), c) integrating the area under each peak in the C(S)distribution to determine the relative concentration of each species ofrecombinant viral particles, d) comparing the amount of recombinantviral particles having an S values corresponding to recombinant viralparticles comprising intact viral genomes to the amount of recombinantviral particles having an S value corresponding to empty capsidparticles, to capsid particles comprising variant recombinant viralgenomes, and/or to the total amount of recombinant viral particles inthe preparation.

In some aspects, the invention provides, methods of monitoring theremoval of empty capsids and/or capsid particles comprising variantrecombinant viral genomes during the purification of a preparation ofrecombinant viral particles, the method comprising removing a sample ofthe recombinant viral particles from the preparation following one ormore steps in the purification process and analyzing the sample for therelative amount of empty capsids and/or capsid particles comprisingvariant recombinant viral genomes according to the method of any one ofclaims 5-8, wherein a decrease in the relative amount of empty capsidsand/or capsids comprising variant genomes to full capsids indicatesremoval of empty capsids from the preparation of recombinant viralparticles. In some embodiments, the presence of a peak that correspondsto the S value of empty capsid particles indicates the presence of emptycapsid particles. In some embodiments, the presence of one or more peaksother than the peak for full capsid particles comprising intactrecombinant viral genomes or empty capsid particles indicates thatpresence of capsid particles comprising variant sized genomes. In someembodiments, the capsid particles comprising variant sized genomescomprise truncated genomes, aggregates, recombinants and/or DNAimpurities.

In some aspects, the invention provides methods of determining theheterogeneity of recombinant viral particles in a preparation ofrecombinant viral particles comprising the steps of a) subjecting thepreparation to analytical ultracentrifugation under boundarysedimentation velocity conditions wherein the sedimentation ofrecombinant viral particles is monitored at time intervals, b) plottingthe differential sedimentation coefficient distribution value (C(s))versus the sedimentation coefficient in Svedberg units (S), wherein thepresence of peaks in addition to the peak representing capsidscomprising an intact viral genome indicates heterogeneity of recombinantparticles in the preparation. In some embodiments, the presence ofadditional peaks indicates the presence of empty capsid particles and/orrecombinant viral particles comprising variant genomes. In someembodiments, the variant genomes are truncated viral genomes,aggregates, recombinants and/or DNA impurities. In some embodiments, themethods further comprise integrating the area under each peak in theC(S) distribution to determine the relative concentration of eachspecies of recombinant viral particles.

In some aspects, the invention provides methods of monitoring thehomogeneity of recombinant viral particles during the purification of apreparation of recombinant viral particles, the method comprisingremoving a sample of the recombinant viral particles from thepreparation following one or more steps in the purification process anddetermining the heterogeneity of recombinant viral particles accordingto the above method, wherein an increase in the relative amount ofrecombinant viral particles comprising intact viral genomes indicates anincrease in the homogeneity of full viral particles in the preparationof recombinant viral particles.

In some embodiments of the above aspects, sedimentation of recombinantviral particles is monitored by absorbance. In some embodiments, theabsorbance is at about 230 nm, 260 nm or 280 nm. In some embodiments,the absorbance is at about 260 nm. In some embodiments, sedimentation ofrecombinant viral particles is monitored by interference. In someembodiments, the interference is Rayleigh interference.

In some embodiments of the above aspects, the preparation is an aqueoussolution. In further embodiments, the aqueous solution comprises apharmaceutical formulation. In some embodiments, the aqueous solutioncomprises a buffer. In some embodiments, the buffer is at physiologicalpH. In some embodiments, the buffer is at physiological osmolality. Insome embodiments, the pharmaceutical formulation comprises phosphatebuffered saline (PBS). In some embodiments, the PBS has pH of about 7.2and an osmolality of about 300 mOsm/L. In some embodiments, themonitoring further comprises comparison to a reference sample, whereinthe reference sample comprises the aqueous solution without recombinantviral particles.

In some embodiments of the above aspects, the C(S) values are determinedby an algorithm that comprises Lamm equation solutions. In someembodiments, the algorithm is the SEDFIT algorithm. In some embodiments,sedimentation is monitored until the recombinant viral particles withthe lowest density sediments to the bottom of a sector of anultracentrifuge; for example, the sector may be a portion of theultracentrifuge comprising a detection system. In some embodiments, theultracentrifugation utilizes an ultracentrifuge comprising anultracentrifuge velocity cell. In some embodiments, sedimentation ismonitored until recombinant viral particles sediment to the bottom ofultracentrifuge velocity cell. In some embodiments, sedimentation ismonitored until the recombinant viral particles with the lowest densitysediments and clears the optical window.

In some embodiments, the radial concentration is recorded for at leastabout any of 0.5 hours, 0.75 hours, 1.0 hours, 1.5 hours, 2.0 hours, 3.0hours, 4.0 hours, or 5.0 hours. In some embodiments, the radialconcentration is recorded for about 1.0 hour. In some embodiments, theradial concentration is recorded for about 1.2 hours. In someembodiments, the radial concentration is recorded from about 0.5 hoursto about 2.0 hours. In some embodiments, the radial concentration isrecorded from about 1.0 hours to about 2.0 hours.

In some embodiments of the above aspects, at least 30 scans are used tomonitor sedimentation of recombinant viral particles. In some aspects,about 30 scans are used to monitor sedimentation of recombinant viralparticles. In other embodiments, about 30 to about 75 scans are used tomonitor sedimentation of recombinant viral particles. In otherembodiments, about 30 to about 50 scans are used to monitorsedimentation of recombinant viral particles. In other embodiments,about 50 to about 75 scans are used to monitor sedimentation ofrecombinant viral particles.

In some embodiments of the above aspects, a regularization is applied toa fitting level with a confidence level of F statistic of at least about0.68. In some embodiments, the regularization is a second derivativeregularization. In some embodiments, the regularization is Max entropyregularization. In some embodiments, the regularization is applied to afitting level with a confidence level of F statistic of about 0.68 toabout 0.90. In some embodiments, the regularization is applied to afitting level with a confidence level of F statistic of about 0.68 toabout 0.99. In some embodiments, the regularization is applied to afitting level with a confidence level of F statistic of about 0.68.

In some embodiments of the above aspects, the following C(S) parametersare held constant: resolution of about 200 S to about 5000 S, S min isabout 1 S to about 100 S, S max is about 100 S to about 5000 S, andfrictional ratio is about 1.0 or is left to float to a value determinedby centrifugation software. In some embodiments, resolution is about 200S to about 10005. In some embodiments, resolution is about 200 S. Insome embodiments, S min is about 1. In some embodiments, Smax is about100 S to about 10005. In other embodiments, Smax is about 200 S to about5000 S. In other embodiments, Smax is about 200 S. In some embodiments,the frictional ratio is left to float to a value determined bycentrifugation software. In some embodiments, the frictional ratio isabout 1.0. In some embodiments, radial invariant (RI) and time invariant(TI) noise subtractions are applied.

In some embodiments of the above aspects, the sedimentation ofrecombinant viral particles is monitored about every 10-60 seconds. Insome embodiments, sedimentation of recombinant viral particles ismonitored (e.g., scanned) about every 10 seconds. In other embodiments,the sedimentation of recombinant viral particles is monitored aboutevery 60 seconds. In some embodiments, the sedimentation velocity ofrecombinant viral during ultracentrifugation is determined by monitoringthe sedimentation of recombinant viral particles once in more than aboutevery 15 seconds, 30 seconds, 45 seconds, 1 minute (60 seconds), 2minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes.

In some embodiments of the above aspects, the boundary sedimentationvelocity is performed at about 3,000 rpm to about 20,000 rpm. In someembodiments, the boundary sedimentation velocity is performed at about3,000 rpm to about 10,000 rpm. In other embodiments, the boundarysedimentation velocity is performed at about 10,000 rpm to about 20,000rpm. In other embodiments, the boundary sedimentation velocity isperformed at about 15,000 rpm to about 20,000 rpm.

In some embodiments of the above aspects, the boundary sedimentationvelocity is performed at about 4° C. to about 20° C. In someembodiments, the boundary sedimentation velocity is performed at about4° C.

In some embodiments of the above aspects, the recombinant viral particleis a recombinant adeno-associated viral (AAV) particle, a recombinantadenovirus particle, a recombinant lentivirus particle or a recombinantherpes simplex viral (HSV) particle. In some embodiments, therecombinant viral particle comprises an AAV1 capsid, an AAV2 capsid, anAAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, anAAV2R471A capsid, an AAV2/2-7m8 capsid, an AAV DJ capsid, an AAV2 N587Acapsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid,a goat AAV capsid, an AAV1/AAV2 chimeric capsid, a bovine AAV capsid, ora mouse AAV capsid rAAV2/HBoV1 (chimeric AAV/human bocavirus virus 1).In some embodiments, the recombinant viral particle comprises an AAV1ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6 ITR, anAAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR, an AAV10 ITR, anAAVrh10 ITR, an AAV11 ITR, or an AAV12 ITR. In some embodiments, the AAVcapsid comprises a tyrosine mutation or a heparin binding mutation. Inother embodiments, the recombinant viral particle is a recombinantadenoviral particle. In some embodiments, the recombinant adenoviralparticle comprises an capsid from Adenovirus serotype 2, 1, 5, 6, 19, 3,11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23,24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35,AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69,bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3. Insome embodiments, the recombinant adenoviral particle comprises avariant of an adenovirus serotype 2 capsid or a variant of an adenoviralserotype 5 capsid. In other embodiments, the recombinant viral particleis a recombinant lentiviral particle. In some embodiments, therecombinant lentiviral particle is pseudotyped with vesicular stomatitisvirus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross river virus(RRV), Ebola virus, Marburg virus, Mokala virus, Rabies virus, RD114 orvariants therein. In other embodiments, the recombinant viral particleis a rHSV particle. In some embodiments, the HSV particle is an HSV-1particle or an HSV-2 particle.

In some aspects, the invention provides methods for evaluating a processfor the production of recombinant viral particles comprising the methodof any one of claims 1 to 67, wherein an increase in the relative amountof recombinant viral particles comprising intact viral genomes comparedto the relative amount of empty capsid particles and/or recombinantviral capsid particles with variant recombinant viral genomes comparedto a reference preparation of recombinant viral particles indicates animprovement in the production of recombinant viral particles. In someembodiments, the recombinant viral particle is a recombinantadeno-associated viral (AAV) particle, a recombinant adenovirusparticle, a recombinant lentivirus particle or a recombinant herpessimplex viral (HSV) particle. In some embodiments, the rAAV particlesare produced from a producer cell line. In other embodiments, the rAAVparticles are produced by triple transfection of i) nucleic acidencoding AAV rep and cap, ii) rAAV vector sequences, and iii) nucleicacid encoding adenovirus helper functions. In other embodiments, therecombinant viral particles are produced by an AAV/HSV hybrid. In otherembodiments, the recombinant viral particles are produced from abaculovirus cell. In some embodiments, the recombinant viral particlesare produced by transient transfection of nucleic acid encoding AAVvector sequences, AAV rep and cap coding regions, and AAV helper virusfunctions to a suitable host cell. In some embodiments, the recombinantviral particles are produced by introduction of one or more nucleicacids encoding AAV vector sequences, AAV rep and cap coding regions, andAAV helper virus functions to a suitable host cell, wherein the one ormore nucleic acids are introduced to the cell using a recombinant helpervirus. In some embodiments, the recombinant helper virus is anadenovirus or a herpes simplex virus. In some embodiments, therecombinant viral particles comprise a self-complementary AAV (scAAV)genome. In some embodiments, the method is used to detect the presenceof recombinant viral particles comprising the monomeric form of a scAAVgenome or the dimeric form of a scAAV genome.

In some embodiments of the above aspect, the recombinant viral particlesare produced by transient transfection of nucleic acid encodingadenovirus vector sequences and adenovirus replication and packagingsequences to a suitable host cell. In other embodiments, the recombinantviral particles are produced by transient transfection of nucleic acidencoding lentivirus vector sequences and/or lentivirus replication andpackaging sequences to a suitable host cell. In other embodiments, therecombinant viral particles are produced by transient transfection ofnucleic acid encoding HSV vector sequences and/or HSV replication andpackaging sequences to a suitable host cell.

In some aspects the invention provides methods for preparing recombinantviral particles with reduced empty capsids and/or recombinant viralparticles comprising variant genomes, the method comprising a) culturinghost cells under conditions suitable for recombinant viral production,wherein the cells comprise i) nucleic acid encoding a heterologoustransgene flanked by at least one AAV ITR, ii) nucleic acid comprisingAAV rep and cap coding regions, wherein the nucleic acid comprises a p5promoter, and iii) nucleic acid encoding AAV helper virus functions; b)lysing the host cells to release recombinant viral particles; c)isolating the recombinant viral particles produced by the host cell; andd) analyzing the recombinant viral particles for the presence of emptycapsids and/or recombinant viral particles with variant genomes byanalytical ultracentrifugation by the above methods. In some aspects theinvention provides methods for preparing recombinant viral particleswith reduced empty capsids and/or recombinant viral particles comprisingvariant genomes, the method comprising a) culturing host cells underconditions suitable for recombinant viral production, wherein the cellscomprise i) nucleic acid encoding a heterologous transgene flanked by atleast one AAV ITR, ii) nucleic acid comprising AAV rep and cap codingregions, wherein the nucleic acid comprises a mutated p5 promoterwherein rep expression from the p5 promoter is reduced compared to awild-type p5 promoter, and iii) nucleic acid encoding AAV helper virusfunctions; b) lysing the host cells to release recombinant viralparticles; c) isolating the recombinant viral particles produced by thehost cell; and d) analyzing the recombinant viral particles for thepresence of empty capsids and/or recombinant viral particles withvariant genomes by analytical ultracentrifugation by the above methods.In some embodiments, the p5 promoter is located 3′ to the rep and/or capcoding region. In some embodiments, the AAV helper virus functionscomprise adenovirus E1A function, adenovirus E1B function, adenovirusE2A function, adenovirus VA function and adenovirus E4 orf6 function.

In some embodiments, of any of the preceding embodiments, therecombinant viral particles have been purified using one or morepurification steps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show that analytical ultracentrifugation (AUC) can beused to characterize recombinant viral vector particles. (FIG. 1A) Arepresentative scanning profile of boundary sedimentation velocitydepicting the absorbance (260 nm) versus the radius (cm) of an AAV2mixture over a time interval (T) of 1.2 hours. The AAV2 mixturecontained empty capsids (“Empty Cap”) and full genome capsids (“IntactVector”). (FIG. 1B) A plot of concentration in units of detection, C(S),versus sedimentation coefficient (Svedberg units, S) showing that AUCcan be used to measure the concentration of empty capsids and fullgenome capsids from an 80%/20% mixture. Each peak is labeled with theparticle species and its corresponding sedimentation coefficient (S) andrelative abundance (%).

FIGS. 2A and 2B show the AUC profiles of pure populations of empty AAV2capsids (FIG. 2A) and genome-containing AAV2-transgene 1 capsids (FIG.2B). Each peak is labeled with the capsid species and its sedimentationcoefficient (S).

FIGS. 3A and 3B show a comparison between interference and absorbancedetection methods by AUC. (FIG. 3A) A plot of differential sedimentationcoefficient distribution value, c(s), vs sedimentation coefficient inSvedberg units (S), yields the distribution of sedimentationcoefficients for a 1:1 mixture of empty and genome-containing capsidsgenerated using interference optical detection. The sedimentationcoefficient and relative abundance (%) for each species are labeled.(FIG. 3B) A plot of differential sedimentation coefficient distributionvalue, c(s), vs sedimentation coefficient in Svedberg units (S), yieldsthe distribution of sedimentation coefficients for a 1:1 mixture ofempty and genome-containing capsids generated using absorbance opticaldetection (260 nm). The sedimentation coefficient and relative abundance(%) for each species are labeled.

FIG. 4 illustrates the triple transfection method for AAV vectorproduction. The three vectors, containing the gene of interest(“pVector”), AAV Rep and Cap genes (“pHLP”), and adenoviral components(“pIAdeno”) are labeled. Note that both genome-containing (labeled withthe “ITR-Transgene-ITR” graphic) and empty capsids (blank) are produced.

FIG. 5 illustrates the producer cell line method for AAV vectorproduction. As labeled, the HeLa S3 cell line contains integrated Rep,Cap, and Puromycin resistance genes, along with an ITR-flanked transgeneof interest. This cell line is infected with adenovirus (“Ad5”) tostimulate recombinant viral production. Note that both genome-containing(labeled “recombinant viral Vector”) and empty capsids are produced, inaddition to adenovirus particles.

FIGS. 6A, 6B and 6C shows that vector production by the producer cellline and triple transfection methods yields different vectorpreparations, as revealed by AUC analysis. (FIG. 6A) A schematic of theAAV2-transgene 2 vector and its 3.4 kb genome. (FIG. 6B) A plot ofdifferential sedimentation coefficient distribution value, c(s), vssedimentation coefficient in Svedberg units (S), yields the distributionof sedimentation coefficients for a vector preparation produced by theproducer cell line method. The sedimentation coefficient and relativeabundance (%) for each species are labeled. (FIG. 6C) A plot ofdifferential sedimentation coefficient distribution value, c(s), vssedimentation coefficient in Svedberg units (S), yields the distributionof sedimentation coefficients for a vector preparation produced by thetriple transfection method. The sedimentation coefficient and relativeabundance (%) for each species are labeled.

FIGS. 7A, 7B and 7C show that the AUC method may be used to monitor thequality and efficacy of vector purification. (FIG. 7A) A plot showingthe purification of full-genome AAV2-transgene 1 capsids from emptycapsids using anion exchange chromatography. Peak fractionscorresponding to each species are labeled. (FIG. 7B) A plot ofdifferential sedimentation coefficient distribution value, c(s), vssedimentation coefficient in Svedberg units (S), yields the distributionof sedimentation coefficients for a vector preparation after elutionfrom the anion exchange column. The sedimentation coefficient andrelative abundance (%) for each species are labeled. (FIG. 7C) A plot ofdifferential sedimentation coefficient distribution value, c(s), vssedimentation coefficient in Svedberg units (S), yields the distributionof sedimentation coefficients for a vector preparation beforechromatography. The sedimentation coefficient and relative abundance (%)for each species are labeled.

FIG. 8 shows the linear relationship between sedimentation coefficientand vector genome size. A standard curve plotting sedimentationcoefficient (S) versus genome size is depicted, along with a line ofbest fit, its formula, and its associated R² value.

FIGS. 9A, 9B and 9C show that assessment of capsid genome size using AUCdata correlates with assessment of genome size by Southern blot. (FIG.9A) A plot of differential sedimentation coefficient distribution value,c(s), vs sedimentation coefficient in Svedberg units (S), yields thedistribution of sedimentation coefficients for a scAAV9 EGFP vectorpreparation. Single stranded monomeric (82 S) and double strandeddimeric (101 S) species are labeled with the corresponding sedimentationcoefficients and relative abundance values (%). A schematic of thevector is also provided. (FIG. 9B) Alkaline Southern blot analysis ofthe DNA from scAAV9 EGFP (lane 1) and single stranded AAV9 EGFP (lane 2)vector capsids. Corresponding bands are labeled as described in the blotlegend. 4.2 and 2.4 kb size standards are provided as labeled. (FIG. 9C)A plot of differential sedimentation coefficient distribution value,c(s), vs sedimentation coefficient in Svedberg units (S), yields thedistribution of sedimentation coefficients for a single stranded AAV9EGFP vector preparation. 82 S and 99 S (full genome) peaks are labeledwith the corresponding sedimentation coefficient and relative abundancevalues (%).

FIGS. 10A, 10B and 10C show that the Rep/Cap promoter position affectsgenome packaging in recombinant viral vectors produced by the tripletransfection method. (FIG. 10A) A schematic of the self-complementaryscAAV2 EGFP vector, with estimated sedimentation coefficients for thedimeric and monomeric genome species. (FIG. 10B) A plot of differentialsedimentation coefficient distribution value, c(s), vs sedimentationcoefficient in Svedberg units (S), yields the distribution ofsedimentation coefficients for a scAAV2 EGFP vector preparation producedusing a “wild-type” helper plasmid with the endogenous p5 promoterdriving Rep 78/68 expression (“WT Rep”). Peaks for single strandedmonomeric (80 S) and double stranded dimeric (100 S) species are labeledwith the corresponding relative abundance values (%). (FIG. 10C) A plotof differential sedimentation coefficient distribution value, c(s), vssedimentation coefficient in Svedberg units (S), yields the distributionof sedimentation coefficients for a scAAV2 EGFP vector preparationproduced using a “wild-type” helper plasmid with the p5 promoter drivingRep 78/68 expression moved downstream of the cap2 sequence (“pHLP Rep”).Peaks for single stranded monomeric (82 S) and double stranded dimeric(100 S) species are labeled with the corresponding relative abundancevalues (%).

FIGS. 11A, 11B, 11C and 11D show that the Rep/Cap promoter positionaffects genome packaging in two additional AAV vectors. (FIGS. 11A and11B) Plots of differential sedimentation coefficient distribution value,c(s), vs sedimentation coefficient in Svedberg units (S), yield thedistribution of sedimentation coefficients for the single stranded AAV5Factor IX vector (AAV5 hFIX16) containing a cap5 sequence produced witha helper plasmid having an endogenous p5 promoter (“WT Rep,” FIG. 11B)or a p5 promoter downstream of the cap5 sequence (“pHLP Rep,” FIG. 11A).(FIGS. 11C-11D) Plots of differential sedimentation coefficientdistribution value, c(s), vs sedimentation coefficient in Svedberg units(S), yield the distribution of sedimentation coefficients for the singlestranded AAV5hSMN vector (AAV5SMN) containing a cap5 sequence producedwith a helper plasmid having an endogenous p5 promoter (“WT Rep,” FIG.11D) or a p5 promoter downstream of the cap5 sequence (“pHLP Rep,” FIG.11C).

FIGS. 12A and 12B reveal that Southern blot analysis correlates with AUCanalysis but misses some fragmented genomes detectable by AUC. (FIG.12A) Southern blot analysis of vector DNA from AAV5SMN preparations madewith the pHLP helper plasmid (lane 2) or the WT Rep plasmid (lane 1).4.6 and 2.4 kb size standards are provided as labeled. (FIG. 12B)Southern blot analysis of vector DNA from AAV5FIX preparations made withthe pHLP helper plasmid (lane 1) or the WT Rep plasmid (lane 2). 4.3,3.0, and 1.9 kb size standards are provided as labeled.

FIG. 13 provides a map of the AAV5 Factor IX vector indicating thepositions of the hFIX transgene, ITR, Rep origin, and AmpR marker gene,among other features. Note that the AmpR marker is upstream of the ITR,enhancer, and promoter region.

FIGS. 14A and 14B show that WT Rep vector genomes, unlike pHLP Repvector genomes, package sequences upstream of the 5′ ITR in the AAV5Factor IX vector. (FIG. 14A) Southern blot analysis using an hFIXtransgene-specific probe comparing pHLP Rep (lane 1) and WT Rep (lane 2)vector genomes. (FIG. 14B) Southern blot analysis using a Repori/AmpR-specific probe comparing pHLP Rep (lane 1) and WT Rep (lane 2)vector genomes.

FIGS. 15A and 15B show the fragmentation of oversized AAV vectorgenomes, as demonstrated by AUC analysis. (FIG. 15A) Plot ofconcentration, C(S), versus sedimentation coefficient (S) generated byAUC for an AAV vector with an oversized genome. This genome contains afull-length chicken β-actin (CBA) promoter driving expression ofβ-phosphodiesterase (ssAAV2/5CBA-βPDE). Peaks for detected species arelabeled by observed sedimentation coefficient (S) and relative abundancevalues (%). (FIG. 15B) Plot of concentration, C(S), versus sedimentationcoefficient (S) generated by AUC for an AAV vector with a truncatedgenome. This genome contains a CBA promoter with a reduced-size introndriving expression of β-phosphodiesterase (AAV5 minCBAPDE6B). Peaks fordetected species are labeled by observed sedimentation coefficient (S)and relative abundance values (%).

FIG. 16 shows the AUC profiles of pure populations of adenoviruscapsids. The sedimentation coefficient (S) and interference values aregiven for each peak.

DETAILED DESCRIPTION

The present invention provides methods of characterizing preparations ofviral particles using analytical ultracentrifugation. By subjectingpreparations to analytical ultracentrifugation (AUC) under boundarysedimentation velocity conditions, the sedimentation of viral particlescan be monitored at time intervals (e.g., one or more times). Thedifferential sedimentation coefficient distribution value (C(s)) versusthe sedimentation coefficient in Svedberg units (S) is then plotted andthe area under each peak in the C(s) distribution is integrated todetermine the relative concentration of each peak. Each peak representsa species of viral particle reflective of its molecular weight. Thespecies that can be detected by these methods include, but are notlimited to, recombinant adeno-associated viral (rAAV) particles,recombinant adenoviral (rAd) particles, recombinant lentiviralparticles, and recombinant herpes simplex viral (rHSV) particles. To userAAV particles as an illustrative example, these methods allow thedetection of rAAV species including rAAV capsid particles comprisingintact rAAV genomes (e.g., full capsids), empty viral capsids wherein norAAV genomes have been encapsidated into viral capsids, and rAAVparticle variants in which variant rAAV genomes are encapsidated inviral capsids (e.g., particles containing AAV-encapsidated DNAimpurities, truncated viral genomes, aggregates, and the like). Thesemethods can be applied to preparations of viral particles regardless ofnucleotide sequence of the viral genome or, in the case of recombinantviral particles, the serotype of the recombinant viral capsid. Thesemethods can be applied to rAAV, rAd, recombinant lentivirus and rHSVviral particles.

In some aspects, the invention provides methods to assess vector genomeintegrity of recombinant viral particles in a preparation of recombinantviral particles by subjecting the preparation to analyticalultracentrifugation under boundary sedimentation velocity conditionswherein the sedimentation of recombinant viral particles is monitored attime intervals (e.g., one or more times). By plotting the differentialsedimentation coefficient distribution value C(S) versus thesedimentation coefficient in Svedberg units (S), species of recombinantviral particles in the preparation can be identified by presence ofpeaks on the plot corresponding to an S value. The genome size of aparticular species of recombinant viral particles can be calculated, forexample, by comparing the S value of the species to a standard curvegenerated by S values of recombinant viral particles comprisingencapsidated viral genomes of different known size. The vector genomesthat can be assessed by these methods include, but are not limited to,recombinant viral capsid particles comprising intact recombinant viralgenomes (e.g., full capsids), empty viral capsids wherein no recombinantviral genomes have been encapsidated into viral capsids, and recombinantviral particle variants in which variant recombinant viral genomes(e.g., particles containing AAV-encapsidated DNA impurities, truncatedviral genomes, aggregates and the like) are encapsidated in viralcapsids. In some embodiments, the viral particles are rAAV, rAd,recombinant lentivirus or rHSV viral particles.

In some embodiments the invention provides methods of determining theheterogeneity of recombinant viral particles (e.g., rAAV, rAd,lentivirus or rHSV particles) in a preparation of recombinant viralparticles by AUC under boundary sedimentation velocity conditionswherein the presence of peaks in a plot of C(S) v. S, in addition to thepeak representing capsids comprising an intact viral genome, indicatesheterogeneity of recombinant viral particles in the preparation. In someembodiments, the relative amounts of each recombinant viral species inthe preparation are calculated by integrating the area for each peak inthe plot.

In some embodiments of the invention, AUC is used to determine thepresence of empty capsids and/or recombinant viral particle variants ina preparation of recombinant viral particles (e.g., rAAV, rAd,lentivirus or rHSV particles), wherein the presence of peak thatcorresponds to the S value of empty capsid particles and/or recombinantviral particle variants in a plot of C(S) vs. S indicates the presenceof empty capsid particles and/or recombinant viral particle variants. Insome embodiments, the relative amount of empty capsids and/orrecombinant viral particle variants in a preparation of recombinantviral particles is determined by integrating the area under each peak ina plot of C(S) versus S and comparing the amount of recombinant viralparticles having an S value corresponding to empty capsid particlesand/or recombinant viral particle variants to the amount of recombinantviral particles having an S value corresponding to recombinant viralparticles comprising intact viral genomes. In some embodiments, theamount of recombinant viral particles having an S value corresponding toempty capsid particles and/or recombinant viral particle variants iscompared to the total amount of all recombinant viral particles in thepreparation by integrating and summing and the area under all the peaksof the plot.

In some embodiments, the invention provides methods of monitoring theremoval of empty capsids and/or recombinant viral particle variantsduring the purification of a preparation of recombinant viral particles(e.g., rAAV, rAd, lentivirus or rHSV particles) by using AUC. Samples ofthe recombinant viral particles from the preparation following one ormore steps in the purification process are analyzed for the relativeamount of empty capsids and/or recombinant viral particle variantswherein a decrease in the relative amount of empty capsids and/orrecombinant viral particle variants to full capsid particles indicatesremoval of empty capsids and/or recombinant viral particle variants fromthe preparation of recombinant viral particles.

In some embodiments, the invention provides methods of evaluatingprocesses for the production of recombinant viral particles (e.g., rAAV,rAd, lentivirus or rHSV particles) by AUC. The preparation ofrecombinant viral particles is analyzed for the presence of intact fullviral capsid particles, empty particles and/or recombinant viralparticle variants. An increase in the relative amount of recombinantviral particles comprising intact viral genomes compared to the relativeamount of empty capsid particles and/or recombinant viral particlevariants (e.g., particles containing AAV-encapsidated DNA impurities,truncated viral genomes, aggregates, and the like) compared to areference preparation of recombinant viral particles (e.g., a standardrecombinant viral preparation process) indicates an improvement in theproduction of recombinant viral particles.

I. General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Molecular Cloning: ALaboratory Manual (Sambrook et al., 4^(th) ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2012); Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., 2003); the series Methodsin Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M. J.MacPherson, B. D. Hames and G. R. Taylor eds., 1995); Antibodies, ALaboratory Manual (Harlow and Lane, eds., 1988); Culture of AnimalCells: A Manual of Basic Technique and Specialized Applications (R. I.Freshney, 6^(th) ed., J. Wiley and Sons, 2010); OligonucleotideSynthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, HumanaPress; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., AcademicPress, 1998); Introduction to Cell and Tissue Culture (J. P. Mather andP. E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: LaboratoryProcedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., J. Wileyand Sons, 1993-8); Handbook of Experimental Immunology (D. M. Weir andC. C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells(J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (C. A.Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: APractical Approach (D. Catty., ed., IRL Press, 1988-1989); MonoclonalAntibodies: A Practical Approach (P. Shepherd and C. Dean, eds., OxfordUniversity Press, 2000); Using Antibodies: A Laboratory Manual (E.Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); TheAntibodies (M. Zanetti and J. D. Capra, eds., Harwood AcademicPublishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J. B. Lippincott Company, 2011).

II. Definitions

A “vector,” as used herein, refers to a recombinant plasmid or virusthat comprises a nucleic acid to be delivered into a host cell, eitherin vitro or in vivo.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. Thus, this term includes, but is not limited to,single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. The backbone of the polynucleotide cancomprise sugars and phosphate groups (as may typically be found in RNAor DNA), or modified or substituted sugar or phosphate groups.Alternatively, the backbone of the polynucleotide can comprise a polymerof synthetic subunits such as phosphoramidates and thus can be anoligodeoxynucleoside phosphoramidate (P—NH₂) or a mixedphosphoramidate-phosphodiester oligomer. In addition, a double-strandedpolynucleotide can be obtained from the single stranded polynucleotideproduct of chemical synthesis either by synthesizing the complementarystrand and annealing the strands under appropriate conditions, or bysynthesizing the complementary strand de novo using a DNA polymerasewith an appropriate primer.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

A “recombinant viral vector” refers to a recombinant polynucleotidevector comprising one or more heterologous sequences (i.e., nucleic acidsequence not of viral origin). In the case of recombinant AAV vectors,the recombinant nucleic acid is flanked by at least one invertedterminal repeat sequence (ITR). In some embodiments, the recombinantnucleic acid is flanked by two inverted terminal repeat sequences(ITRs).

A “recombinant AAV vector (recombinant adeno-associated viral vector)”refers to a polynucleotide vector comprising one or more heterologoussequences (i.e., nucleic acid sequence not of AAV origin) that areflanked by at least one AAV inverted terminal repeat sequences (ITR). Insome embodiments, the recombinant nucleic acid is flanked by twoinverted terminal repeat sequences (ITRs). Such recombinant viralvectors can be replicated and packaged into infectious viral particleswhen present in a host cell that has been infected with a suitablehelper virus (or that is expressing suitable helper functions) and thatis expressing AAV rep and cap gene products (i.e. AAV Rep and Capproteins). When a recombinant viral vector is incorporated into a largerpolynucleotide (e.g., in a chromosome or in another vector such as aplasmid used for cloning or transfection), then the recombinant viralvector may be referred to as a “pro-vector” which can be “rescued” byreplication and encapsidation in the presence of AAV packaging functionsand suitable helper functions. A recombinant viral vector can be in anyof a number of forms, including, but not limited to, plasmids, linearartificial chromosomes, complexed with lipids, encapsulated withinliposomes, and encapsidated in a viral particle, for example, an AAVparticle. A recombinant viral vector can be packaged into an AAV viruscapsid to generate a “recombinant adeno-associated viral particle(recombinant viral particle)”.

An “rAAV virus” or “rAAV viral particle” refers to a viral particlecomposed of at least one AAV capsid protein and an encapsidated rAAVvector genome.

A “recombinant adenoviral vector” refers to a polynucleotide vectorcomprising one or more heterologous sequences (i.e., nucleic acidsequence not of adenovirus origin) that are flanked by at least oneadenovirus inverted terminal repeat sequence (ITR). In some embodiments,the recombinant nucleic acid is flanked by two inverted terminal repeatsequences (ITRs). Such recombinant viral vectors can be replicated andpackaged into infectious viral particles when present in a host cellthat is expressing essential adenovirus genes deleted from therecombinant viral genome (e.g., E1 genes, E2 genes, E4 genes, etc.).When a recombinant viral vector is incorporated into a largerpolynucleotide (e.g., in a chromosome or in another vector such as aplasmid used for cloning or transfection), then the recombinant viralvector may be referred to as a “pro-vector” which can be “rescued” byreplication and encapsidation in the presence of adenovirus packagingfunctions. A recombinant viral vector can be in any of a number offorms, including, but not limited to, plasmids, linear artificialchromosomes, complexed with lipids, encapsulated within liposomes, andencapsidated in a viral particle, for example, an adenovirus particle. Arecombinant viral vector can be packaged into an adenovirus virus capsidto generate a “recombinant adenoviral particle.”

A “recombinant lentivirus vector” refers to a polynucleotide vectorcomprising one or more heterologous sequences (i.e., nucleic acidsequence not of lentivirus origin) that are flanked by at least onelentivirus terminal repeat sequences (LTRs). In some embodiments, therecombinant nucleic acid is flanked by two lentiviral terminal repeatsequences (LTRs). Such recombinant viral vectors can be replicated andpackaged into infectious viral particles when present in a host cellthat has been infected with a suitable helper functions. A recombinantlentiviral vector can be packaged into a lentivirus capsid to generate a“recombinant lentiviral particle.”

A “recombinant herpes simplex vector (recombinant HSV vector)” refers toa polynucleotide vector comprising one or more heterologous sequences(i.e., nucleic acid sequence not of HSV origin) that are flanked by HSVterminal repeat sequences. Such recombinant viral vectors can bereplicated and packaged into infectious viral particles when present ina host cell that has been infected with a suitable helper functions.When a recombinant viral vector is incorporated into a largerpolynucleotide (e.g., in a chromosome or in another vector such as aplasmid used for cloning or transfection), then the recombinant viralvector may be referred to as a “pro-vector” which can be “rescued” byreplication and encapsidation in the presence of HSV packagingfunctions. A recombinant viral vector can be in any of a number offorms, including, but not limited to, plasmids, linear artificialchromosomes, complexed with lipids, encapsulated within liposomes, andencapsidated in a viral particle, for example, an HSV particle. Arecombinant viral vector can be packaged into an HSV capsid to generatea “recombinant herpes simplex viral particle.”

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is compared or into which itis introduced or incorporated. For example, a polynucleotide introducedby genetic engineering techniques into a different cell type is aheterologous polynucleotide (and, when expressed, can encode aheterologous polypeptide). Similarly, a cellular sequence (e.g., a geneor portion thereof) that is incorporated into a viral vector is aheterologous nucleotide sequence with respect to the vector.

The term “transgene” refers to a polynucleotide that is introduced intoa cell and is capable of being transcribed into RNA and optionally,translated and/or expressed under appropriate conditions. In aspects, itconfers a desired property to a cell into which it was introduced, orotherwise leads to a desired therapeutic or diagnostic outcome. Inanother aspect, it may be transcribed into a molecule that mediates RNAinterference, such as siRNA.

The terms “genome particles (gp),” “genome equivalents,” or “genomecopies” as used in reference to a viral titer, refer to the number ofvirions containing the recombinant viral DNA genome or RNA genome,regardless of infectivity or functionality. The number of genomeparticles in a particular vector preparation can be measured byprocedures such as described in the Examples herein, or for example, inClark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al.(2002) Mol. Ther., 6:272-278.

The terms “infection unit (iu),” “infectious particle,” or “replicationunit,” as used in reference to a viral titer, refer to the number ofinfectious and replication-competent recombinant viral vector particlesas measured by the infectious center assay, also known as replicationcenter assay, as described, for example with AAV, in McLaughlin et al.(1988) J. Virol., 62:1963-1973.

The term “transducing unit (tu)” as used in reference to a viral titer,refers to the number of infectious recombinant viral vector particlesthat result in the production of a functional transgene product asmeasured in functional assays such as described in Examples herein, orfor example regarding AAV, in Xiao et al. (1997) Exp. Neurobiol.,144:113-124; or in Fisher et al. (1996) J. Virol., 70:520-532 (LFUassay).

An “inverted terminal repeat” or “ITR” sequence is a term wellunderstood in the art and refers to relatively short sequences found atthe termini of viral genomes which are in opposite orientation.

An “AAV inverted terminal repeat (ITR)” sequence, a term well-understoodin the art, is an approximately 145-nucleotide sequence that is presentat both termini of the native single-stranded AAV genome. The outermost125 nucleotides of the ITR can be present in either of two alternativeorientations, leading to heterogeneity between different AAV genomes andbetween the two ends of a single AAV genome. The outermost 125nucleotides also contains several shorter regions ofself-complementarity (designated A, A′, B, B′, C, C′ and D regions),allowing intrastrand base-pairing to occur within this portion of theITR.

A “terminal resolution sequence” or “trs” is a sequence in the D regionof the AAV ITR that is cleaved by AAV rep proteins during viral DNAreplication. A mutant terminal resolution sequence is refractory tocleavage by AAV rep proteins.

“AAV helper functions” refer to functions that allow AAV to bereplicated and packaged by a host cell. AAV helper functions can beprovided in any of a number of forms, including, but not limited to,helper virus or helper virus genes which aid in AAV replication andpackaging. Other AAV helper functions are known in the art such asgenotoxic agents.

A “helper virus” for AAV refers to a virus that allows AAV (which is adefective parvovirus) to be replicated and packaged by a host cell. Ahelper virus provides “helper functions” which allow for the replicationof AAV. A number of such helper viruses have been identified, includingadenoviruses, herpesviruses, poxviruses such as vaccinia andbaculovirus. The adenoviruses encompass a number of different subgroups,although Adenovirus type 5 of subgroup C (Ad5) is most commonly used.Numerous adenoviruses of human, non-human mammalian and avian origin areknown and are available from depositories such as the ATCC. Viruses ofthe herpes family, which are also available from depositories such asATCC, include, for example, herpes simplex viruses (HSV), Epstein-Banviruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV).Examples of adenovirus helper functions for the replication of AAVinclude E1A functions, E1B functions, E2A functions, VA functions andE4orf6 functions. Baculoviruses available from depositories includeAutographa californica nuclear polyhedrosis virus.

A preparation of rAAV is said to be “substantially free” of helper virusif the ratio of infectious AAV particles to infectious helper virusparticles is at least about 10²:1; at least about 10⁴:1, at least about10⁶:1; or at least about 10⁸:1 or more. In some embodiments,preparations are also free of equivalent amounts of helper virusproteins (i.e., proteins as would be present as a result of such a levelof helper virus if the helper virus particle impurities noted above werepresent in disrupted form). Viral and/or cellular protein contaminationcan generally be observed as the presence of Coomassie staining bands onSDS gels (e.g., the appearance of bands other than those correspondingto the AAV capsid proteins VP1, VP2 and VP3).

As used herein, “differential coefficient distribution value” or “C(S)”is a variant of the distribution of Lamm equation solutions to describedistributions of sedimenting particles; for example duringultracentrifugation.

As used herein, “Svedberg units” refers to a unit for sedimentationrate. The sedimentation rate for a particle of a given size and shapemeasures how fast the particle sediments. One Svedberg unit isequivalent to 10⁻¹³ seconds. For example, Svedberg units are often usedto reflect the rate at which a molecule travels under the centrifugalforce of a centrifuge.

As used herein, “sedimentation velocity conditions” or “boundarysedimentation velocity conditions” may refer to any experimentalconditions under which a sample solution is subjected to sedimentationvelocity analysis. Sedimentation velocity allows the study of particlesover a wide range of pH and ionic strength conditions and attemperatures 4 to 40° C. The rate at which the sedimentation boundarymoves is a measure of the sedimentation coefficient of the sedimentingspecies. The sedimentation coefficient depends on the molecular weight(larger particles sediment faster) and also on molecular shape. Theminimum width of the sedimentation boundary is related to the diffusioncoefficient of the molecule; the presence of multiple species withsimilar sedimentation coefficients will cause the boundary to be broaderthan expected on the basis of diffusion alone. Sedimentation velocityconditions may include without limitation any conditions related to therotor speed, distance between sample and rotor center, temperature,solvent, sample, buffer, ultracentrifugation time, time interval fordetection, sector and optical window characteristics, AUCinstrumentation (including ultracentrifuge and detection apparatus),equilibrium dialysis of reference solvent, and data analysis algorithms.

As used herein, the term “analytical density gradient sedimentationequilibrium” relates to methods for measuring the buoyant density of aparticle, or using differences in buoyant density to separate differentspecies of particles. These methods may use, for example, AUCsedimentation equilibrium techniques. In these methods, a particlesolution (e.g., without limitation, a solution of a polypeptide,polynucleotide, or viral capsids) may be subjected toultracentrifugation in a gradient solvate, such as a cesium chloride orcesium sulfate gradient, until equilibrium with the solvate is attained.At equilibrium, the particle solution will concentrate, or band, at theposition in the gradient where the density of the particle is equal tothat of the solvate. The position of bands may be used to calculateparticle density, or a band may be extracted to isolate a single speciesof particle.

As used herein, the “SEDFIT algorithm” is an algorithm that allows oneto analyze hydrodynamic data such as sedimentation velocity (Schuck(2000) Biophys. J., 78:1606-19). In the SEDFIT algorithm, a grid ofsedimentation coefficients across an expected range is created.Sedimentation boundaries are simulated using solutions to the Lammequation for each sedimentation coefficient, assuming constant particleshape and solvent frictional ratio.

As used herein, the term “F statistic” or “F ratio” refers to theconfidence level. This parameter controls the amount of regularizationused. It has a different meaning for different ranges: From 0 to 0.5, noregularization is used. Values from 0.5 to 0.999 correspond toprobabilities P (confidence levels). From these P-values, the desiredchi-square increase allowed for the parsimony constraint of theregularization is calculated with F-statistics. A value of 0.51 willcause very little regularization; values of 0.68 to 0.90 wouldcorrespond to commonly used confidence levels (usually, with 50 scans ormore the chi-square increase corresponding to a probability of 0.7 is ofthe order of 0.1%), while values close to 0.99 would cause very highregularization. The relationship of these values with probabilities canbe examined using the F-statistics calculator. If numbers >1 areentered, they are taken directly as chi-square ratios (as there are noprobabilities >1). For example, a value of 1.1 will result inregularization with 10% chi-square increase.

To “reduce” is to decrease, reduce or arrest an activity, function,and/or amount as compared to a reference. In certain embodiments, by“reduce” is meant the ability to cause an overall decrease of 20% orgreater. In another embodiment, by “reduce” is meant the ability tocause an overall decrease of 50% or greater. In yet another embodiment,by “reduce” is meant the ability to cause an overall decrease of 75%,85%, 90%, 95%, or greater.

A “reference” as used herein, refers to any sample, standard, or levelthat is used for comparison purposes. For example, when measuringabsorbance or refraction of AAV in an aqueous solution, the absorbanceor refraction of the solution is compared to the absorbance orrefraction of the aqueous solution without AAV (i.e. a referencesolution). In other examples, a reference may refer to a standardprocedure known in the art. For example, when analyzing a procedure forimproved quality of AAV production (e.g., homogeneity), the AAV producedby the candidate procedure is compared to procedures known in the art(i.e. reference procedures).

An “isolated” molecule (e.g., nucleic acid or protein) or cell means ithas been identified and separated and/or recovered from a component ofits natural environment. Thus, for example, isolated rAAV particles maybe prepared using a purification technique to enrich it from a sourcemixture, such as a culture lysate or production culture supernatant.Enrichment can be measured in a variety of ways, such as, for example,by the proportion of DNase-resistant particles (DRPs) present in asolution, or by infectivity, or it can be measured in relation to asecond, potentially interfering substance present in the source mixture,such as contaminants, including production culture contaminants orin-process contaminants, including helper virus, media components, andthe like, as defined below.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.”

As used herein, the singular form of the articles “a,” “an,” and “the”includes plural references unless indicated otherwise. For example, thephrase “a rAAV particle” includes one or more rAAV particles.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and/or “consistingessentially of” aspects and embodiments.

III. Analytical Ultracentrifugation

Analytical ultracentrifugation is a means to evaluate the molecularweight and the hydrodynamic and thermodynamic properties of a protein orother macromolecule. Heterogeneity of a protein or macromolecule bysedimentation velocity over a range of conditions includingconcentration, temperature, ionic strength, and pH. For example, aprotein may be analyzed in a clinically relevant formulation. Use ofanalytical ultracentrifugation to characterize adenovirus preparationsis provided by Berkowitz, S A & Philo J S, (2007) Anal. Biochem.,362:16-37.

In certain aspects, the present invention provides methods ofcharacterizing preparations of viral particles using analyticalultracentrifugation (AUC). For example, in some embodiments, theinvention provides methods to assess vector genome integrity ofrecombinant adeno-associated viral (rAAV) particles in preparations ofrAAV particles using AUC to distinguish viral particles with full,intact genomes, empty viral capsids and viral particles with variant(e.g., truncated, aggregates, impurities and the like) viral genomes. Inothers embodiments, these methods may be applied in a similar way toanalyze adenovirus, lentivirus, and herpes simplex virus (HSV)particles. AUC analysis refers to quantitative methods forcharacterizing the biophysical properties of particles (e.g.,polypeptides, polynucleotides, and viral capsids) by measuring theirmigration through a solvent in a centrifugal field. AUC analysis hasbeen well characterized over many decades and is highly versatile.Because AUC analysis relies upon first-principle hydrodynamic andthermodynamic information, AUC may be applied to determine thebiophysical properties of many types of particles across a wide range ofparticle concentrations and sizes. AUC analysis typically encompassestwo basic types of experiment: sedimentation velocity and sedimentationequilibrium. Sedimentation equilibrium analysis yields thermodynamicproperties of particles that may be used to measure characteristics suchas stoichiometry and association constants. Sedimentation velocityyields hydrodynamic properties of particles that may be used to measurecharacteristics such as size, shape, and concentration. A feature of AUCanalysis of viral preparations is that the same assay conditions may beused to analyze different preparations of viral particles regardless ofnucleotide sequence of the viral genome or serotype of the capsid.

Certain aspects of the present disclosure relate to the use ofsedimentation velocity analysis to characterize viral capsid properties.In some embodiments, sedimentation velocity analysis uses anultracentrifuge velocity cell with two sectors in dialysis equilibrium(one for an experimental sample and one for a solvent-only referencesample), each containing two optical windows that allow light to passthrough the compartment. Ultracentrifugation applies an angular velocityto the cell and leads to rapid sedimentation of the solute particlestowards the bottom of the sector. As sedimentation occurs, solute isdepleted near the meniscus at the top of the cell, creating asedimenting boundary between the depleted region and the sedimentingsolute. The rate of movement or migration of the sedimenting boundary ismeasured by taking measurements that compare the properties of thesample and reference sectors at specific time intervals (forsedimentation velocity, these intervals are typically on the order ofminutes). If multiple species of solute are present, this may lead tothe formation of multiple sedimenting boundaries, each corresponding toa resolvable species.

Several methods for optically detecting a sedimenting boundary andmeasuring its rate of movement or migration are known in the art (forreference, see Cole et al. (2008) Methods Cell Biol., 84:143-79). Insome embodiments, the reference and sample sectors may be assayed usingabsorbance detection. In this detection method, the absorbance at aparticular wavelength may be measured for the sample and referencesectors at different radial positions within each sector. Alternatively,the time course of absorbance at a single radial position may bemeasured. Beer's Law provides a mathematical relationship betweenabsorbance and a solute's extinction coefficient.

In some embodiments, the reference and sample sectors may be assayedusing interference detection (e.g., Rayleigh interference detection). Inthe Rayleigh interference detection method, the interference opticalsystem contains two parallel slits. A single, coherent beam of light issplit such that it passes through both windows, and then the two beamsare re-merged. When these two light waves are merged, they form aninterference pattern of alternating light and dark fringes. If thesample and reference samples were to have an identical refractive index,the resulting interference fringes would be perfectly straight.Increasing the concentration of solute increases the solution'srefractive index, thereby retarding the sample light beam and causing avertical fringe shift. By measuring this fringe shift, one may measurethe concentration of solute in the sample. Unlike absorbance detection,which measures absolute values for the sample and reference,interference detection measures a relative difference between the sampleand reference. However, interference detection yields integrated peaksthat are directly proportional to concentration, and it may be used fortypes of samples that do not absorb significantly. For a reference onusing Rayleigh interference optics with AUC, see Furst (1997) Eur.Biophys. J. 35:307-10.

Measurement of the rate at which the sedimentation boundary moves may beused to derive many physical properties of solute particles. The rate ofthe boundary movement determines the sedimentation coefficient, which isbased on the mass and shape (frictional coefficient) of the particle.The sedimentation coefficient of a particle, s, refers to the ratio ofits velocity to the acceleration applied to it by a centrifugal field.Sedimentation coefficients are expressed in Svedberg units, S (oneSvedberg unit is equivalent to 10⁻¹³ seconds). The sedimentationcoefficient of a particle or solution of particles depends upon itsproperties, for example molecular weight (corrected for buoyancy), andthe properties of the solvent.

The change in the concentration boundary of a solute over time duringultracentrifugation may be determined using the Lamm equation (Schuck(2000) Biophys. J., 78:1606-19). Briefly, the Lamm equation calculatesthe change in the concentration boundary of a solute over time inresponse to the competing forces of sedimentation (which concentratesthe solute) and diffusion (which disperses the solute), taking intoaccount the sector-shaped cell and the centrifugal field generated bythe rotor. The Lamm equation may be expressed as:∂c/∂t=D[(∂{circumflex over ( )}2c/∂r{circumflex over( )}2)+1/r(∂c/∂r)]−sω{circumflex over ( )}2[r(∂c/∂r)+2c]  Equation 1:where c is the solute concentration, D represents the solute diffusionconstant, s represents the sedimentation coefficient, ω represents theangular velocity of the rotor, r is the radius, and t is time.

By fitting raw AUC data to solutions of the Lamm equation, it ispossible to determine solute characteristics such as the sedimentationcoefficient and the change in concentration distribution. For example,experimentally determined values for the rate of change of a sedimentingboundary may be modeled using the Lamm equation to derive thesedimentation coefficient, molecular mass, or concentration of thesolute forming the boundary. Several programs known in the art, such asSEDFIT (Schuck (2000) Biophys. J., 78:1606-19), may be used to model theLamm equation to AUC data. These programs are also able to apply theLamm equation to solutions containing multiple solutes or multiplesedimenting boundaries.

One example of a suitable program for the determination of solutecharacteristics is the SEDFIT algorithm. In some embodiments, the SEDFITalgorithm may be used to calculate a differential coefficientdistribution value, or C(S), using AUC data from a solution containing amixture of particle species (for reference, see Schuck (2000) Biophys.J., 78:1606-19). In the SEDFIT algorithm, a grid of sedimentationcoefficients across an expected range is created.

Sedimentation boundaries are simulated using solutions to the Lammequation for each sedimentation coefficient, assuming constant particleshape and solvent frictional ratio. Actual AUC data are then fit tothese Lamm solutions to derive the differential coefficient distributionvalue, or C(S). Many other programs useful for analyzing AUC data may befound in Cole and Hansen (1999) J. Biomol. Tech. 10:163-76.

In some embodiments of the invention, recombinant viral particles arehighly purified, suitably buffered, and concentrated. In someembodiments, the viral particles are concentrated to at least about anyof 1×10⁷ vg/mL, 2×10⁷ vg/mL, 3×10⁷ vg/mL, 4×10⁷ vg/mL, 5×10⁷ vg/mL,6×10⁷ vg/mL, 7×10⁷ vg/mL, 8×10⁷ vg/mL, 9×10⁷ vg/mL, 1×10⁸ vg/mL, 2×10⁸vg/mL, 3×10⁸ vg/mL, 4×10⁸ vg/mL, 5×10⁸ vg/mL, 6×10⁸ vg/mL, 7×10⁸ vg/mL,8×10⁸ vg/mL, 9×10⁸ vg/mL, 1×10⁹ vg/mL, 2×10⁹ vg/mL, 3×10⁹ vg/mL, 4×10⁹vg/mL, 5×10⁹ vg/mL, 6×10⁹ vg/mL, 7×10⁹ vg/mL, 8×10⁹ vg/mL, 9×10⁹ vg/mL,1×10¹⁰ vg/mL, 2×10¹⁰ vg/mL, 3×10¹⁰ vg/mL, 4×10¹⁰ vg/mL, 5×10¹⁰ vg/mL,6×10¹⁰ vg/mL, 7×10¹⁰ vg/mL, 8×10¹⁰ vg/mL, 9×10¹⁰ vg/mL, 1×10¹¹ vg/mL,2×10¹¹ vg/mL, 3×10¹¹ vg/mL, 4×10¹¹ vg/mL, 5×10¹¹ vg/mL, 6×10¹¹ vg/mL,7×10¹¹ vg/mL, 8×10¹¹ vg/mL, 9×10¹¹ vg/mL, 1×10¹² vg/mL, 2×10¹² vg/mL,3×10¹² vg/mL, 4×10¹² vg/mL, 5×10¹² vg/mL, 6×10¹² vg/mL, 7×10¹² vg/mL,8×10¹² vg/mL, 9×10¹² vg/mL, 1×10¹³ vg/mL, 2×10¹³ vg/mL, 3×10¹³ vg/mL,4×10¹³ vg/mL, 5×10¹³ vg/mL, 6×10¹³ vg/mL, 7×10¹³ vg/mL, 8×10¹³ vg/mL,9×10¹³ vg/mL. In some embodiments, the viral particles are concentratedto of about 1×10⁷ vg/mL to about 1×10¹³ vg/mL, about 1×10⁸ vg/mL toabout 1×10¹³ vg/mL, about 1×10⁹ vg/mL to about 1×10¹³ vg/mL, about1×10¹⁰ vg/mL to about 1×10¹³ vg/mL, about 1×10¹¹ vg/mL to about 1×10¹³vg/mL, about 1×10¹² vg/mL to about 1×10¹³ vg/mL, about 1×10⁷ vg/mL toabout 1×10¹² vg/mL, about 1×10⁸ vg/mL to about 1×10¹² vg/mL, about 1×10⁹vg/mL to about 1×10¹² vg/mL, about 1×10¹⁰ vg/mL to about 1×10¹² vg/mL,about 1×10¹¹ vg/mL to about 1×10¹² vg/mL, about 1×10⁷ vg/mL to about1×10¹¹ vg/mL, about 1×10⁸ vg/mL to about 1×10¹¹ vg/mL, about 1×10⁹ vg/mLto about 1×10¹¹ vg/mL, about 1×10¹⁰ vg/mL to about 1×10¹¹ vg/mL, about1×10⁷ vg/mL to about 1×10¹⁰ vg/mL, about 1×10⁸ vg/mL to about 1×10¹⁰vg/mL, about 1×10⁹ vg/mL to about 1×10¹⁰ vg/mL, about 1×10⁷ vg/mL toabout 1×10⁹ vg/mL, about 1×10⁸ vg/mL to about 1×10⁹ vg/mL, or about1×10⁷ vg/mL to about 1×10⁸ vg/mL.

In some embodiments, viral particles are generated in a suitable hostcells and purified. In some embodiments, the viral particles arepurified by affinity chromatography. Methods to purify viral particles(e.g., AAV particles, adenovirus particles, lentivirus particles, HSVparticles) are known in the art. For example, by use of an antibody of aviral capsid protein or binding ligand of a viral capsid proteinimmobilized on a chromatography media. Examples of viral capsid affinitychromatographies include but are not limited to AVB affinitychromatography for AAV (GE Healthcare), metal affinity chromatographyfor adenovirus and HSV, and heparin affinity chromatography for AAV andlentivirus, and the like. Methods to purify adenovirus particles arefound, for example, in Bo, H et al., (2014) Eur. J. Pharm. Sci.67C:119-125. Methods to purify lentivirus particles are found, forexample, in Segura M M, et al., (2013) Expert Opin Biol Ther.13(7):987-1011. Methods to purify HSV particles are found, for example,in Goins, W F et al., (2014) Herpes Simplex Virus Methods in MolecularBiology 1144:63-79.

In some embodiments, the recombinant viral particles are formulated in apharmaceutical composition. In related embodiments, the pharmaceuticalcomposition contains a buffer having physiological pH and/orphysiological osmolality. A nonlimiting example of a pharmaceuticalformulation is phosphate buffered saline (PBS) and in some embodiments,the PBS can be at physiological osmolality (e.g., about pH 7.2 and about300 mOsm/L). In some embodiments, sample adjustments are made to targetconcentration by optical density measurement at 260 nm from 0.1 to 1.0.In some examples, this concentration results in reproducible andconsistent AUC data. In some examples, concentration of viral particlesis adjusted either by direct dilution with PBS or further concentration;for example, by using a centrifugal filter device.

In some embodiments of the invention, sedimentation velocity analyticalultracentrifugation (SV-AUC) analysis is performed using an analyticalultracentrifuge that is capable of characterizing a sample in its nativestate under biologically relevant solution conditions (e.g.,ProteomeLab™ XL-I (Beckman Coulter)). When using the ProteomeLab™ XL-1,sample is loaded into the sample sector of a two sector velocity cell, avehicle control (e.g., PBS without recombinant viral) is loaded into thecorresponding reference sector. The sample is placed in the four-holerotor and allowed to equilibrate in the instrument until a temperatureof about 20° C. and full vacuum are maintained for about one hour. In anexemplary embodiment, sedimentation velocity centrifugation is performedat about 20,000 RPM, about 20° C., and about 0.003 cm radial stepsetting, with no delay and with no replicates. As noted below, differentparameters may be used for centrifugation. In some embodiments,absorbance (260 nm) and/or interference optics (e.g., Rayleighinterference optics) are used to simultaneously record radialconcentration as a function of time until the smallest sedimentingcomponent clears the optical window. In some embodiments, the radialconcentration is recorded until the sedimenting species with the lowestdensity clears the sector. In some embodiments, sedimentation ismonitored until the recombinant viral particles with the lowest densitysediments to the bottom of a sector of an ultracentrifuge. A sector maybe a portion of an ultracentrifuge; for example an ultracentrifugevelocity cell. In some embodiments, a sector may be a portion of anultracentrifuge where samples are detected. In some embodiments, theultracentrifugation utilizes an ultracentrifuge comprising anultracentrifuge velocity cell. In some embodiments, is monitored untilrecombinant viral particles sediment to the bottom of an ultracentrifugevelocity cell. In some embodiments, sedimentation is monitored until therecombinant viral particles with the lowest density sediments and clearsthe optical window. In some embodiments, the radial concentration isrecorded for at least about any of 0.5 hours, 0.75 hours, 1.0 hours, 1.5hours, 2.0 hours, 3.0 hours, 4.0 hours, or 5.0 hours. In someembodiments, the radial concentration is recorded for between any ofabout 0.5 hours to about 0.75 hours, about 0.75 hours to about 1.0hours, about 1.0 hours to about 1.5 hours, about 1.5 hours to about 2.0hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours,about 4 hours to about 5 hours. In some embodiments, the radialconcentration is recorded for about 1.2 hours. Optimizing runsconditions may include, for example, continuing the run until all of thesedimenting species are fully sedimented to the bottom of the sector,with the temperature held constant at 20° C. and a speed between 18,000rpm and 20,000 rpm. As noted below, other temperatures and speeds may beused.

The percent full capsid is determined by analyzing a multiple of scans(e.g., 75) from each detection method using the SEDFIT continuous sizeC(S) distribution model. Second (2^(nd)) derivative regularization isapplied to the fitting. In some embodiments, the confidence level of Fstatistic is about 0.68. In some embodiments, the confidence level of Fstatistic is more than about any of 0.68, 0.70, 0.75, 0.80, 0.85, 0.90,0.95 or 0.99. In some embodiments, the confidence level of F statisticis about 0.68 to about 0.90. In some embodiments, the confidence levelof F statistic is about 0.68 to about 0.99. In some embodiments, thefollowing C(S) parameters are held constant: resolution of about 200 Sto about 5000 S, S min is about 1 S to about 100 S, S max is about 100 Sto about 5000 S, and frictional ratio is about 1.0 or is left to floatto a value determined by centrifugation software. In some embodiments,the resolution is about any of 200 S, 300 S, 400 S, 500 S, 600 S, 700 S,800 S, 900 S, or 1000 S. In some embodiments, the resolution is betweenany of about 200 S to about 1000 S, 200 S to about 900 S, 200 S to about800 S, 200 S to about 700 S, 200 S to about 600 S, 200 S to about 500 S,200 S to about 400 S, 200 S to about 300 S, 300 S to about 1000 S, 300 Sto about 900 S, 300 S to about 800 S, 300 S to about 700 S, 300 S toabout 600 S, 300 S to about 500 S, 300 S to about 400 S, 400 S to about1000 S, 400 S to about 900 S, 400 S to about 800 S, 400 S to about 700S, 400 S to about 600 S, 400 S to about 500 S, 500 S to about 1000 S,500 S to about 900 S, 500 S to about 800 S, 500 S to about 700 S, 500 Sto about 600 S, 600 S to about 1000 S, 600 S to about 900 S, 600 S toabout 800 S, 600 S to about 700 S, 700 S to about 1000 S, 700 S to about900 S, 700 S to about 800 S, 800 S to about 1000 S, 800 S to about 900S, or 900 S to about 1000 S. In some embodiments, the resolution isabout 200 S. In some embodiments, the Smax is about any of 100 S, 200 S,300 S, 400 S, 500 S, 600 S, 700 S, 800 S, 900 S, or 1000 S. In someembodiments, the Smax is between any of about 100 S to about 1000 S, 100S to about 900 S, 100 S to about 800 S, 100 S to about 700 S, 100 S toabout 600 S, 100 S to about 500 S, 100 S to about 400 S, 100 S to about300 S, 100 S to about 200 S, 200 S to about 1000 S, 200 S to about 900S, 200 S to about 800 S, 200 S to about 700 S, 200 S to about 600 S, 200S to about 500 S, 200 S to about 400 S, 200 S to about 300 S, 300 S toabout 1000 S, 300 S to about 900 S, 300 S to about 800 S, 300 S to about700 S, 300 S to about 600 S, 300 S to about 500 S, 300 S to about 400 S,400 S to about 1000 S, 400 S to about 900 S, 400 S to about 800 S, 400 Sto about 700 S, 400 S to about 600 S, 400 S to about 500 S, 500 S toabout 1000 S, 500 S to about 900 S, 500 S to about 800 S, 500 S to about700 S, 500 S to about 600 S, 600 S to about 1000 S, 600 S to about 900S, 600 S to about 800 S, 600 S to about 700 S, 700 S to about 1000 S,700 S to about 900 S, 700 S to about 800 S, 800 S to about 1000 S, 800 Sto about 900 S, or 900 S to about 1000 S. In some embodiments, Smax isabout 200 S to about 5000 S. In some embodiments, wherein Smax is about200 S. In some embodiments, the frictional ratio is left to float to avalue determined by centrifugation software. In some embodiments, thefrictional ratio is about 1.0. In some embodiments, radial invariant(RI) and time invariant (TI) noise subtractions are applied. In someembodiments, the meniscus position is allowed to float, letting thesoftware choose the optimal position. In some embodiments, thefrictional ratio is allowed to float, letting the software choose theoptimal position. The model fits the data to the Lamm equation, and theresulting size distribution is a “distribution of sedimentationcoefficients” that looks like a chromatogram with the area under eachpeak proportional to concentration in units of Fringes or OD₂₆₀ units.The sedimentation coefficient (in Svedberg units) and the relativeconcentration (in OD units) are determined for each component in thedistribution. In some embodiments, multiple AUC runs are independentassays, and each analysis the following attributes are monitored toensure quality of results: goodness of fit (rmsd), the ratio of OD₂₆₀interference signal in fringes (A260/IF ratio) for each peak,consistency of sedimentation coefficients for each species between runs,and overall quality of the scans.

In some embodiments of the invention, extinction coefficients are usedto calculate molar concentration and the actual percent value of theintact vector peak from absorbance data. Molar absorbance extinctioncoefficients for both empty capsids (€_(260/capsid)=3.72e6) and intactvector (€_(260/vector)=3.00e7) can be calculated based on publishedformulae (Sommer et al. (2003) Mol Ther., 7:122-8). Extinctioncoefficients are available for empty capsid and intact vector peaks. TheC(S) values can be determined using the SEDFIT algorithm described bySchuck (2000) Biophys. J., 78:1606-19. Molar concentration of bothintact vector and empty capsid can be calculated using Beer's Law andthe percentage of full capsid are calculated from these values. In someembodiments, values are reported in terms of the percentage of fullcapsid.

In some embodiments, it is not possible to determine empirically theextinction coefficient of particular species of recombinant viralparticles (e.g., viral particles with fragmented genomes of unknown sizeand sequence). A relationship between S value and genome size may beestablished by analyzing recombinant viral vector preps withencapsidated viral genomes of known nucleotide size and a correspondingS value are determined as described herein. The calculated S values canbe plotted to generate a standard curve to which recombinant viralspecies of unknown molecular weight or genome size can be compared todetermine the molecular weight of the unknown species.

In some aspects, the invention provides methods of characterizing apreparation of recombinant viral particles (e.g., rAAV, rAd, lentiviral,or rHSV particles) comprising the steps of a) subjecting the preparationto analytical ultracentrifugation under boundary sedimentation velocityconditions wherein the sedimentation of recombinant viral particles ismonitored at time intervals (e.g., one or more times), b) plotting thedifferential sedimentation coefficient distribution value (C(s)) versusthe sedimentation coefficient in Svedberg units (S), c) integrating thearea under each peak in the C(s) distribution to determine the relativeconcentration of each peak, wherein each peak represents a species ofrecombinant viral particle. In some embodiments, the species ofrecombinant viral particle identified by the methods of the inventioninclude, but are not limited to; full recombinant viral particlescomprising intact recombinant viral genomes, empty recombinant viralcapsid particles, and recombinant viral particles comprising variantrecombinant viral genomes. In some embodiments the variant genomes aresmaller than the intact recombinant viral genome (e.g., truncatedgenomes). In some embodiments, the variant genomes are larger than theintact recombinant viral genome (e.g., aggregates, recombinants, etc.).In some embodiments, the invention provides methods to assess vectorgenome integrity of recombinant viral particles in a preparation ofrecombinant viral particles comprising a) subjecting the preparation toanalytical ultracentrifugation under boundary sedimentation velocityconditions wherein the sedimentation of recombinant viral particles ismonitored at time intervals (e.g., one or more times), b) plotting thedifferential sedimentation coefficient distribution value C(s) versusthe sedimentation coefficient in Svedberg units (S), c) identifyingspecies of recombinant viral particles in the preparation by presence ofpeaks on the plot corresponding to an S value, wherein the genome sizeof a particular species of recombinant viral particles is calculated bycomparing the S value of the species to a standard curve generated by Svalues of recombinant viral particles comprising encapsidated viralgenomes of different known size. In some embodiments, the methodsfurther comprise integrating the area under each peak in the C(S)distribution to determine the relative concentration of each species ofrecombinant viral particles. In some embodiments, the sedimentation ofrecombinant viral particles is monitored at one time interval. In someembodiments, the sedimentation of recombinant viral particles ismonitored at more than one time interval.

In some embodiments of the invention, the sedimentation of recombinantviral particles (e.g., rAAV, rAd, lentiviral, or rHSV particles) ismonitored by measuring optical density or absorbance at about 260 nmMeans of measuring absorbance are known in the art. In some embodiments,an ultracentrifuge used for AUC is equipped with means for measuringabsorbance. In other embodiments, the sedimentation of recombinant viralparticles is monitored by interference. In some embodiments, thesedimentation of recombinant viral particles is monitored by Rayleighinterference. Means of measuring interference are known in the art(Furst (1997) Eur. Biophys. J. 35:307-10). In some embodiments, anultracentrifuge used for AUC is equipped with means for measuringinterference. In some embodiments, the sedimentation of recombinantviral particles is monitored by both absorbance and interference. Insome embodiments, the absorbance and/or interference are measured usinga reference standard. In some embodiments, the reference standardmatches the solution of the recombinant viral preparation with theexception that the recombinant viral is not present. For example, therecombinant viral preparation may comprise recombinant viral in a buffersuch as phosphate buffered saline. In this example, the referencestandard may be phosphate buffered saline without recombinant viralparticles.

In some embodiments of the invention, the preparation of viral particlesis in a pharmaceutical formulation. Such formulations are well known inthe art (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition,pp. 1035-1038 and 1570-1580). Such pharmaceutical formulations can besterile liquids, such as water and oil, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, and the like. Saline solutions and aqueous dextrose,polyethylene glycol (PEG) and glycerol solutions can also be employed asliquid carriers, particularly for injectable solutions. Thepharmaceutical formulation may further comprise additional ingredients,for example preservatives, buffers, tonicity agents, antioxidants andstabilizers, nonionic wetting or clarifying agents, viscosity-increasingagents, and the like. In some embodiments of the invention thepharmaceutical formulation comprises phosphate buffered saline.

In some embodiments of the invention, the sedimentation velocity ofviral particles during ultracentrifugation is determined by monitoringthe sedimentation of viral particles continuously duringultracentrifugation. It is within the purview of the skilled artisan tooptimize the parameters of AUC for different types of viral particles.Without wishing to be bound to theory, a range of AUC settings thatallows the analysis of both AAV and adenovirus particles should enablethe analysis of other viral particles including lentivirus and HSV sincethe size of HSV and lentiviral particles is between that of AAV andadenovirus particles. In some embodiments, data acquisition for rAAV,rHSV, lentiviral, and/or rAd particles is performed with an AUC speed ofbetween about 3,000 and about 20,000 rpm. In some embodiments, dataanalysis for rAAV, HSV, lentiviral, and/or adenoviral particles isperformed with an S_(min) of about 1 S and an S_(max) of about 10005. Insome embodiments, data analysis for rAAV, rHSV, lentiviral, and/or rAdparticles is performed with a resolution of about 200 S to about 1,000S. In some embodiments, the resolution is about any of 200 S, 300 S, 400S, 500 S, 600 S, 700 S, 800 S, 900 S, or 10005. In some embodiments, theresolution is between any of about 200 S to about 10005, 200 S to about900 S, 200 S to about 800 S, 200 S to about 700 S, 200 S to about 600 S,200 S to about 500 S, 200 S to about 400 S, 200 S to about 300 S, 300 Sto about 1000 S, 300 S to about 900 S, 300 S to about 800 S, 300 S toabout 700 S, 300 S to about 600 S, 300 S to about 500 S, 300 S to about400 S, 400 S to about 1000 S, 400 S to about 900 S, 400 S to about 800S, 400 S to about 700 S, 400 S to about 600 S, 400 S to about 500 S, 500S to about 1000 S, 500 S to about 900 S, 500 S to about 800 S, 500 S toabout 700 S, 500 S to about 600 S, 600 S to about 1000 S, 600 S to about900 S, 600 S to about 800 S, 600 S to about 700 S, 700 S to about 1000S, 700 S to about 900 S, 700 S to about 800 S, 800 S to about 1000 S,800 S to about 900 S, or 900 S to about 1000 S. In some embodiments, theresolution is about 200 S. data analysis for rAAV, rHSV, lentiviral,and/or rAd particles is performed with an Smax of about any of 100 S,200 S, 300 S, 400 S, 500 S, 600 S, 700 S, 800 S, 900 S, or 1000 S. Insome embodiments, the Smax is between any of about 100 S to about 1000S, 100 S to about 900 S, 100 S to about 800 S, 100 S to about 700 S, 100S to about 600 S, 100 S to about 500 S, 100 S to about 400 S, 100 S toabout 300 S, 100 S to about 200 S, 200 S to about 1000 S, 200 S to about900 S, 200 S to about 800 S, 200 S to about 700 S, 200 S to about 600 S,200 S to about 500 S, 200 S to about 400 S, 200 S to about 300 S, 300 Sto about 1000 S, 300 S to about 900 S, 300 S to about 800 S, 300 S toabout 700 S, 300 S to about 600 S, 300 S to about 500 S, 300 S to about400 S, 400 S to about 1000 S, 400 S to about 900 S, 400 S to about 800S, 400 S to about 700 S, 400 S to about 600 S, 400 S to about 500 S, 500S to about 1000 S, 500 S to about 900 S, 500 S to about 800 S, 500 S toabout 700 S, 500 S to about 600 S, 600 S to about 1000 S, 600 S to about900 S, 600 S to about 800 S, 600 S to about 700 S, 700 S to about 1000S, 700 S to about 900 S, 700 S to about 800 S, 800 S to about 1000 S,800 S to about 900 S, or 900 S to about 1000 S. In some embodiments,Smax is about 200 S to about 5000 S. In some embodiments, wherein Smaxis about 200 S. In some embodiments, radial invariant (RI) and timeinvariant (TI) noise subtractions are applied. In some embodiments, themeniscus position is allowed to float, letting the software choose theoptimal position. In some embodiments, the frictional ratio is allowedto float, letting the software choose the optimal position. In someembodiments, data analysis for rAAV and/or adenoviral particles is heldconstant at 1. In some embodiments, data analysis for rAAV, HSV,lentiviral, and/or adenoviral particles is allowed to float by using theFIT command with a value optimized using non-linear regression.

With respect to recombinant viral particles (e.g., rAAV, rAd,lentiviral, or rHSV particles), in some embodiments, the sedimentationvelocity of recombinant viral during ultracentrifugation is determinedby monitoring (e.g., scanning) the sedimentation of recombinant viralparticles once in more than about every 15 seconds, 30 seconds, 45seconds, 1 minute (60 seconds), 2 minutes, 3 minutes, 4 minutes, 5minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes. Scans may be continuously acquiredwithout delay as quickly as the optical systems allow. Interferencescans are rapid, and a single scan is complete in ˜10-15 seconds, whileabsorbance scans require ˜60 seconds. When dual detection is used thespeed of scan acquisition for both are determined by the absorbancesystem. In some embodiments of the invention, more than about 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100scans are used to monitor sedimentation of recombinant viral particlesduring ultracentrifugation. In some embodiments, a minimum of 30 scansis required for analysis, and scans are collected until thesedimentation process is complete. In some embodiments, thesedimentation process may typically be described by between 40 and 75scans. In some embodiments, the sedimentation velocity of recombinantviral particles is determined based on about 75 scans. In someembodiments, the sedimentation velocity of recombinant viral particlesis determined based on about 55 scans to about 75 scans. In someembodiments, the sedimentation velocity of recombinant viral particlesis determined based on about 55 scans to about 60 scans. In someembodiments, the sedimentation velocity of recombinant viral particlesis determined based on about 60 scans to about 75 scans. In someembodiments, the sedimentation velocity of recombinant viral particlesis determined based on about 60 scans to about 70 scans. In someembodiments, the sedimentation velocity of recombinant viral particlesis determined based on multiple ultracentrifugations (runs). In someembodiments, the sedimentation velocity of recombinant viral particlesis determined based on any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreultracentrifugation runs. In some embodiments, the sedimentationvelocities are used to determine C(S) values using the SEDFIT algorithm.In some embodiments, a second derivative regularization is applied to afitting level with a confidence level of F statistic of about 0.68. Insome embodiments, the following C(S) parameters are held constant:resolution 100 S to about 200 S, S min is about 1, S max is about 200 Sto 300 S, and frictional ratio is about 1.0 to 1.2 S. In someembodiments, radial invariant (RI) and time invariant (TI) noisesubtractions are applied.

In some embodiments of the invention, the boundary sedimentationvelocity of recombinant viral particles (e.g., rAAV, rAd, lentiviral, orrHSV particles) in a preparation of recombinant viral particles byultracentrifuging the preparation of recombinant viral particles at morethan about any of 5,000 rpm; 10,000 rpm; 15,000 rpm; 20,000 rpm; 25,000rpm; 30,000 rpm; 35,000 rpm; 40,000 rpm; 45,000 rpm; or 50,000 rpm. Insome embodiments, the ultracentrifugation is run at between any of about5,000 rpm and about 50,000 rpm; about 10,000 rpm and about 50,000 rpm;about 15,000 rpm and about 50,000 rpm; about 20,000 rpm and about 50,000rpm; about 25,000 rpm and about 50,000 rpm; about 30,000 rpm and about50,000 rpm; about 35,000 rpm and about 50,000 rpm; about 40,000 rpm andabout 50,000 rpm; about 45,000 rpm and about 50,000 rpm; about 5,000 rpmand about 45,000 rpm; about 10,000 rpm and about 45,000 rpm; about15,000 rpm and about 45,000 rpm; about 20,000 rpm and about 45,000 rpm;about 25,000 rpm and about 45,000 rpm; about 30,000 rpm and about 45,000rpm; about 40,000 rpm and about 45,000 rpm; about 5,000 rpm and about40,000 rpm; about 10,000 rpm and about 40,000 rpm; about 15,000 rpm andabout 40,000 rpm; about 20,000 rpm and about 40,000 rpm; about 25,000rpm and about 40,000 rpm; about 30,000 rpm and about 40,000 rpm; about35,000 rpm and about 40,000 rpm; about 5,000 rpm and about 35,000 rpm;about 10,000 rpm and about 35,000 rpm; about 15,000 rpm and about 35,000rpm; about 20,000 rpm and about 35,000 rpm; about 25,000 rpm and about35,000 rpm; about 30,000 rpm and about 35,000 rpm; about 5,000 rpm andabout 30,000 rpm; about 10,000 rpm and about 30,000 rpm; about 15,000rpm and about 30,000 rpm; about 20,000 rpm and about 30,000 rpm; about25,000 rpm and about 30,000 rpm; about 5,000 rpm and about 25,000 rpm;about 10,000 rpm and about 25,000 rpm; about 20,000 rpm and about 25,000rpm; about 5,000 rpm and about 20,000 rpm; about 10,000 rpm and about20,000 rpm; about 15,000 rpm and about 20,000 rpm; about 5,000 rpm andabout 15,000 rpm; about 10,000 rpm and about 15,000 rpm; or about 5,000rpm and about 10,000 rpm. In some embodiments of the invention, theboundary sedimentation velocity of recombinant viral particles in apreparation of recombinant viral particles by ultracentrifuging thepreparation of recombinant viral particles at about 20,000 rpm. In someembodiments of the invention, the boundary sedimentation velocity ofrecombinant viral particles in a preparation of recombinant viralparticles by ultracentrifuging the preparation of recombinant viralparticles at about 15,000 rpm to about 20,000 rpm.

In some embodiments of the invention, the boundary sedimentationvelocity of recombinant viral particles in a preparation of recombinantviral particles (e.g., rAAV, rAd, lentiviral, or rHSV particles) byultracentrifuging the preparation of recombinant viral particles atabout or more than 4° C., 10° C., 15° C., 20° C., 25° C., or 30° C. Insome embodiments, the ultracentrifugation is run as between any of about4° C. and about 30° C., about 4° C. and about 25° C., about 4° C. andabout 20° C., about 4° C. and about 15° C., about 4° C. and about 10°C., about 10° C. and about 30° C., about 10° C. and about 25° C., about10° C. and about 20° C., about 10° C. and about 15° C., about 15° C. andabout 30° C., about 15° C. and about 25° C., about 15° C. and about 20°C., about 20° C. and about 30° C., or about 20° C. and about 25° C. Insome embodiments, the boundary sedimentation velocity of recombinantviral particles in a preparation of recombinant viral particles byultracentrifuging the preparation of recombinant viral particles atabout 20° C. In some embodiments, the boundary sedimentation velocity ofrecombinant viral particles in a preparation of recombinant viralparticles by ultracentrifuging the preparation of recombinant viralparticles at about 15° C. to about 20° C.

As disclosed herein, numerous types of recombinant viral particles maybe analyzed by the methods of the present disclosure (e.g., AAV,adenoviral, lentiviral, and/or HSV particles). Suitableultracentrifugation conditions, analysis algorithms, and otherparameters may be determined empirically through methods known in theart. Exemplary parameters for AAV, adenoviral, lentiviral, and HSVparticles, along with guidance for the selection of specific parameteroptions, are provided without limitation in Table 1 below.

TABLE 1 Exemplary parameters for AAV, adenoviral, lentiviral, and HSVparticles. AAV Ad Lentivirus HSV Exemplary buffers Phosphate basedPhosphate based Phosphate based Phosphate based buffer at buffer atbuffer at buffer at physiologic pH, and physiologic pH, physiologic pH,physiologic pH, physiologic and physiologic and physiologic andphysiologic osmolality osmolality osmolality osmolality ~300 mOsM/L ~300mOsM/L L ~300 mOsM/L ~300 mOsM/L Exemplary algorithms for Any algorithmAny algorithm Any algorithm Any algorithm determining C(S) using Lammusing Lamm using Lamm using Lamm equation solutions; equation solutions;equation solutions; equation solutions; e.g., SEDFIT C(S) e.g., SEDFITC(S) e.g., SEDFIT C(S) e.g., SEDFIT C(S) Exemplary number of 30-99930-999 30-999 30-999 scans (minimum, *excess scans can maximum, ranges)always be collected and then excluded from analysis (such as skip everyother scan)- scans that occur after complete sedimentation of virus canbe excluded Exemplary confidence F = 0.68 F = 0.68 F = 0.68 F = 0.68level of the F statistic Exemplary ranges for 1-100S 1-100S 1-100S1-100S Smin Exemplary ranges for 100-1000S 100-5000S 100-5000S 100-5000SSmax Exemplary ranges for *Resolution 200-5000S 200-5000S 200-5000Sresolution depends on S Max 200S-1000S Exemplary frictional Use the FITUse FIT command Use FIT command Use FIT command ratio command to todetermine to determine to determine determine frictional frictionalratio or frictional ratio frictional ratio ratio. Since AAV is set at 1.~spherical, in embodiments, 1 may be used as the frictional ratio.Exemplary ranges for 10,000-20,000 rpm 3,000-10,000 rpm 3,000-10,000 rpm3,000-10,000 rpm AUC speed Exemplary absorbances 260 nm 260 nm 260 nm260 nm for monitoring 280 nm 280 nm 280 nm 280 nm sedimentation of viral230 nm 230 nm 230 nm 230 nm particles Exemplary methods for IF IF IF IFmonitoring sedimentation Absorbance Absorbance Absorbance Absorbance ofviral particles Radial invariant (RI) and Ti and RI noise Ti and RInoise Ti and RI noise Ti and RI noise time invariant (TI) noisecorrection required correction correction correction requiredsubtractions, alternative for interference required for required for forinterference subtractions/calculations detection. interferenceinterference detection. May or may not be detection. detection. May ormay not be used with May or may not be May or may not be used withabsorbance used with used with absorbance detection absorbanceabsorbance detection detection detection Scanning frequency *when usingScan with no Scan with no Scan with no absorbance delay through scandelay through scan delay through scan detection system, with 60 secondwith 60 second with 60 second limited by speed of delay delay delayabsorbance scan (~60 Seconds)-scan as fast as system allows with nodelay IF only: collect every 10-60 seconds (10-60 second delay)Temperature ranges 4° C.-20° C. 4° C.-20° C. 4° C.-20° C. 4° C.-20° C.

In some aspects, the invention provides methods to determine thepresence of empty capsids in a preparation of recombinant viralparticles (e.g., rAAV, rAd, lentiviral, or rHSV particles) comprisingthe steps of a) subjecting the preparation to analyticalultracentrifugation under boundary sedimentation velocity conditionswherein the sedimentation of recombinant viral particles is monitored attime intervals (e.g., one or more times), b) plotting the differentialsedimentation coefficient distribution value (C(s)) versus thesedimentation coefficient in Svedberg units (S), wherein the presence ofpeak that corresponds to the S value of empty capsid particles indicatesthat presence of empty capsid particles. In some embodiments, theinvention provides methods of measuring the relative amount emptycapsids in a preparation of recombinant viral particles comprising thesteps of a) subjecting the preparation to analytical ultracentrifugationunder boundary sedimentation velocity conditions wherein thesedimentation of recombinant viral particles is monitored at timeintervals (e.g., one or more times), b) plotting the differentialsedimentation coefficient distribution value (C(s)) versus thesedimentation coefficient in Svedberg units (S), c) integrating the areaunder each peak in the C(S) distribution to determine the relativeconcentration of each species of recombinant viral particles, d)comparing the amount of recombinant viral particles having an S valuecorresponding to empty capsid particles to the amount of recombinantviral particles having an S value corresponding to recombinant viralparticles comprising intact viral genomes. In some embodiments, theamount of recombinant viral particles having an S value corresponding toempty capsid particles is compared to the total amount of allrecombinant viral particles in the preparation by integrating all peakson the plot of C(S) vs. S.

In some aspects, the invention provides methods to determine thepresence of recombinant viral particle variants in a preparation ofrecombinant viral particles (e.g., rAAV, rAd, lentiviral, or rHSVparticles) comprising the steps of a) subjecting the preparation toanalytical ultracentrifugation under boundary sedimentation velocityconditions wherein the sedimentation of recombinant viral particles ismonitored at time intervals (e.g., one or more times), b) plotting thedifferential sedimentation coefficient distribution value (C(s)) versusthe sedimentation coefficient in Svedberg units (S), wherein thepresence of peak that corresponds to the S value that differs from the Svalue of recombinant viral capsid particles comprising a full intactrecombinant viral genome indicates that presence of recombinant viralparticle variants. In some embodiments, the invention provides methodsof measuring the relative amount recombinant viral particle variants ina preparation of recombinant viral particles comprising the steps of a)subjecting the preparation to analytical ultracentrifugation underboundary sedimentation velocity conditions wherein the sedimentation ofrecombinant viral particles is monitored at time intervals (e.g., one ormore times), b) plotting the differential sedimentation coefficientdistribution value (C(s)) versus the sedimentation coefficient inSvedberg units (S), c) integrating the area under each peak in the C(S)distribution to determine the relative concentration of each species ofrecombinant viral particles, d) comparing the amount of recombinantviral particles having an S value corresponding to empty capsidparticles to the amount of recombinant viral particles having an S valuecorresponding to recombinant viral particles comprising intact viralgenomes. In some embodiments, the amount of recombinant viral particleshaving an S value that differs from the S value of recombinant viralcapsid particles comprising a full intact recombinant viral genome iscompared to the total amount of all recombinant viral particles in thepreparation by integrating all peaks on the plot of C(S) vs. S. In someembodiments, the recombinant viral particle variants compriserecombinant viral genomes that are smaller (e.g., truncated) or largerthan the full length intact viral genome. Other viral-encapsidated DNAimpurities can also be detected.

In some embodiments, the invention provides methods of monitoring theremoval of empty capsids and/or recombinant viral particles with variantgenomes during the purification of a preparation of recombinant viralparticles (e.g., rAAV, rAd, lentiviral, or rHSV particles) the methodcomprising removing a sample of the recombinant viral particles from thepreparation following one or more steps in the purification process andanalyzing the sample for the relative amount of empty capsids using AUCas described herein. A decrease in the relative amount of empty capsidsand/or recombinant viral particles comprising variant genomes to fullcapsids indicates removal of empty capsids from the preparation ofrecombinant viral particles.

In some embodiments, the invention provides methods of determining theheterogeneity of recombinant viral particles in a preparation ofrecombinant viral particles (e.g., rAAV, rAd, lentiviral, or rHSVparticles) comprising the steps of a) subjecting the preparation toanalytical ultracentrifugation under boundary sedimentation velocityconditions wherein the sedimentation of recombinant viral particles ismonitored at time intervals (e.g., one or more times), b) plotting thedifferential sedimentation coefficient distribution value (C(s)) versusthe sedimentation coefficient in Svedberg units (S), wherein thepresence of peaks in addition to the peak representing capsidscomprising an intact viral genome indicates heterogeneity of recombinantviral particles in the preparation. In some embodiments, the species ofrecombinant viral particle identified by the methods of the inventioninclude, but are not limited to full recombinant viral particlescomprising intact recombinant viral genomes, empty recombinant viralcapsid particles, and recombinant viral particles comprising variantrecombinant viral genomes. In some embodiments the variant genomes aresmaller than the intact recombinant viral genome (e.g., truncatedgenomes). In some embodiments, the variant genomes are larger than theintact recombinant viral genome (e.g., aggregates, recombinants, etc.).In some embodiments the variant genomes include genomes that are smallerand larger than the intact recombinant viral genome.

In some embodiments, the invention provides methods of monitoring theheterogeneity of recombinant viral particles during the purification ofa preparation of recombinant viral particles (e.g., rAAV, rAd,lentiviral, or rHSV particles) the method comprising removing a sampleof the recombinant viral particles from the preparation following one ormore steps in the purification process and determining the relativeamount of full capsids comprising an intact recombinant viral genome,empty capsids and/or recombinant viral particles with variant genomesusing AUC as described herein, wherein an increase in the relativeamount of recombinant viral particles comprising intact viral genomesindicates an increase in the homogeneity of full viral particles in thepreparation of recombinant viral particles.

In embodiments of the embodiments described above, the recombinant viralparticles have been purified using one or more purification steps.Examples of purification steps include but are not limited toequilibrium centrifugation, anion exchange filtration, tangential flowfiltration (TFF), apatite chromatography, heat inactivation of helpervirus, hydrophobic interaction chromatography, immunoaffinitychromatography, size exclusion chromatography (SEC), nanofiltration,cation exchange chromatography, and anion exchange chromatography.

In embodiments of the embodiments described above, the recombinant viralparticles comprise a self-complementary AAV (scAAV) genome. In someembodiments, the recombinant AAV genome comprises a first heterologouspolynucleotide sequence (e.g., a therapeutic transgene coding strand)and a second heterologous polynucleotide sequence (e.g., the noncodingor antisense strand of the therapeutic transgene) wherein the firstheterologous polynucleotide sequence can form intrastrand base pairswith the second polynucleotide sequence along most or all of its length.In some embodiments, the first heterologous polynucleotide sequence anda second heterologous polynucleotide sequence are linked by a sequencethat facilitates intrastrand basepairing; e.g., a hairpin DNA structure.Hairpin structures are known in the art, for example in siRNA molecules.In some embodiments, the first heterologous polynucleotide sequence anda second heterologous polynucleotide sequence are linked by a mutatedITR. In some embodiments, the scAAV viral particles comprise a monomericform of an scAAV genome. In some embodiments, the scAAV viral particlescomprise the dimeric form of and scAAV genome. In some embodiments, AUCas described herein is used to detect the presence of rAAV particlescomprising the monomeric form of an scAAV genome. In some embodiments,AUC as described herein is used to detect the presence of rAAV particlescomprising the dimeric form of an scAAV genome. In some embodiments, thepackaging of scAAV genomes into capsid is monitored by AUC describedherein.

In embodiments of the embodiments described above, the rAAV particlescomprise an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid,an AAV5 capsid, an AAV6 capsid (e.g., a wild-type AAV6 capsid, or avariant AAV6 capsid such as ShH10, as described in U.S. PG Pub.2012/0164106), an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, anAAVrh8R, an AAV9 capsid (e.g., a wild-type AAV9 capsid, or a modifiedAAV9 capsid as described in U.S. PG Pub. 2013/0323226), an AAV10 capsid,an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, a tyrosine capsidmutant, a heparin binding capsid mutant, an AAV2R471A capsid, anAAVAAV2/2-7m8 capsid, an AAV DJ capsid (e.g., an AAV-DJ/8 capsid, anAAV-DJ/9 capsid, or any other of the capsids described in U.S. PG Pub.2012/0066783), an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708Acapsid, an AAV V708K capsid, a goat AAV capsid, an AAV1/AAV2 chimericcapsid, a bovine AAV capsid, a mouse AAV capsid, or an AAV capsiddescribed in U.S. Pat. No. 8,283,151 or International Publication No.WO/2003/042397. In embodiments of the above embodiments described above,the rAAV particles comprise at least one AAV1 ITR, AAV2 ITR, AAV3 ITR,AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR,AAV10 ITR, AAVrh10 ITR, AAV11 ITR, AAV12 ITR, AAV DJ ITR, goat AAV ITR,bovine AAV ITR, or mouse AAV ITR. In some embodiments, the rAAVparticles comprise ITRs from one AAV serotype and AAV capsid fromanother serotype. For example, the rAAV particles may comprise atherapeutic transgene flanked by at least one AAV2 ITR encapsidated intoan AAV9 capsid. Such combinations may be referred to as pseudotyped rAAVparticles.

IV. Viral Particles

The methods disclosed herein may find use, inter alia, in characterizingspecies of interest in a variety of viral particles (e.g., viralparticles with a full genome, as compared to viral particles withtruncated genomes and/or viral particles comprising DNA impurities).

In some embodiments, the viral particle is a recombinant AAV particlecomprising a nucleic acid comprising a transgene flanked by one or twoITRs. The nucleic acid is encapsidated in the AAV particle. The AAVparticle also comprises capsid proteins. In some embodiments, thenucleic acid comprises the protein coding sequence(s) of interest (e.g.,a therapeutic transgene) operatively linked components in the directionof transcription, control sequences including transcription initiationand termination sequences, thereby forming an expression cassette. Theexpression cassette is flanked on the 5′ and 3′ end by at least onefunctional AAV ITR sequences. By “functional AAV ITR sequences” it ismeant that the ITR sequences function as intended for the rescue,replication and packaging of the AAV virion. See Davidson et al., PNAS,2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; andPechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporatedherein in their entirety by reference. For practicing some aspects ofthe invention, the recombinant vectors comprise at least all of thesequences of AAV essential for encapsidation and the physical structuresfor infection by the rAAV. AAV ITRs for use in the vectors of theinvention need not have a wild-type nucleotide sequence (e.g., asdescribed in Kotin, Hum. Gene Ther., 1994, 5:793-801), and may bealtered by the insertion, deletion or substitution of nucleotides or theAAV ITRs may be derived from any of several AAV serotypes. More than 40serotypes of AAV are currently known, and new serotypes and variants ofexisting serotypes continue to be identified. See Gao et al., PNAS,2002, 99(18): 11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; andBossis et al., J. Virol., 2003, 77(12):6799-810. Use of any AAV serotypeis considered within the scope of the present invention. In someembodiments, a rAAV vector is a vector derived from an AAV serotype,including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11, AAV12, a tyrosine capsid mutant, aheparin binding capsid mutant, an AAV2R471A capsid, an AAVAAV2/2-7m8capsid, an AAV DJ capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, anAAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1/AAV2chimeric capsid, a bovine AAV capsid, or a mouse AAV capsid, or thelike. In some embodiments, the nucleic acid in the AAV comprises an ITRof AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8,AAVrh10, AAV11, AAV12 or the like. In further embodiments, the rAAVparticle comprises capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11, AAV12 or the like. Infurther embodiments, the rAAV particle comprises capsid proteins of anAAV serotype from Clades A-F (Gao, et al. J. Virol. 2004, 78(12):6381).

Different AAV serotypes are used to optimize transduction of particulartarget cells or to target specific cell types within a particular targettissue (e.g., a diseased tissue). A rAAV particle can comprise viralproteins and viral nucleic acids of the same serotype or a mixedserotype. For example, a rAAV particle can comprise AAV9 capsid proteinsand at least one AAV2 ITR or it can comprise AAV2 capsid proteins and atleast one AAV9 ITR. In yet another example, a rAAV particle can comprisecapsid proteins from both AAV9 and AAV2, and further comprise at leastone AAV2 ITR. Any combination of AAV serotypes for production of a rAAVparticle is provided herein as if each combination had been expresslystated herein.

In some embodiments, the AAV comprises at least one AAV1 ITR and capsidprotein from any of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAVrh.8, AAVrh10, AAV11, and/or AAV12. In some embodiments, the AAVcomprises at least one AAV2 ITR and capsid protein from any of AAV1,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11,and/or AAV12. In some embodiments, the AAV comprises at least one AAV3ITR and capsid protein from any of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, and/or AAV12. In some embodiments,the AAV comprises at least one AAV4 ITR and capsid protein from any ofAAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11,and/or AAV12. In some embodiments, the AAV comprises at least one AAV5ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV6, AAV7,AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, and/or AAV12. In some embodiments,the AAV comprises at least one AAV6 ITR and capsid protein from any ofAAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11,and/or AAV12. In some embodiments, the AAV comprises at least one AAV7ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, and/or AAV12. In some embodiments,the AAV comprises at least one AAV8 ITR and capsid protein from any ofAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAVrh.8, AAVrh10, AAV11,and/or AAV12. In some embodiments, the AAV comprises at least one AAV9ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAVrh.8, AAVrh10, AAV11, and/or AAV12. In some embodiments,the AAV comprises at least one AAVrh8 ITR and capsid protein from any ofAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV11, and/orAAV12. In some embodiments, the AAV comprises at least one AAVrh10 ITRand capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV11, and/or AAV12. In some embodiments, the AAV comprisesat least one AAV11 ITR and capsid protein from any of AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAVrh10, and/or AAV12. Insome embodiments, the AAV comprises at least one AAV12 ITR and capsidprotein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAVrh10, and/or AAV11.

Self-Complementary AAV Viral Genomes

In some aspects, the invention provides viral particles comprising arecombinant self-complementing genome. AAV viral particles withself-complementing genomes and methods of use of self-complementing AAVgenomes are described in U.S. Pat. Nos. 6,596,535; 7,125,717; 7,765,583;7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z.,et al., (2003) Gene Ther 10:2105-2111, each of which are incorporatedherein by reference in its entirety. A rAAV comprising aself-complementing genome will quickly form a double stranded DNAmolecule by virtue of its partially complementing sequences (e.g.,complementing coding and non-coding strands of a transgene). In someembodiments, the invention provides an AAV viral particle comprising anAAV genome, wherein the rAAV genome comprises a first heterologouspolynucleotide sequence (e.g., a therapeutic transgene coding strand)and a second heterologous polynucleotide sequence (e.g., the noncodingor antisense strand of the therapeutic transgene) wherein the firstheterologous polynucleotide sequence can form intrastrand base pairswith the second polynucleotide sequence along most or all of its length.In some embodiments, the first heterologous polynucleotide sequence anda second heterologous polynucleotide sequence are linked by a sequencethat facilitates intrastrand basepairing; e.g., a hairpin DNA structure.Hairpin structures are known in the art, for example in siRNA molecules.In some embodiments, the first heterologous polynucleotide sequence anda second heterologous polynucleotide sequence are linked by a mutatedITR (e.g., the right ITR). The mutated ITR comprises a deletion of the Dregion comprising the terminal resolution sequence. As a result, onreplicating an AAV viral genome, the rep proteins will not cleave theviral genome at the mutated ITR and as such, a recombinant viral genomecomprising the following in 5′ to 3′ order will be packaged in a viralcapsid: an AAV ITR, the first heterologous polynucleotide sequenceincluding regulatory sequences, the mutated AAV ITR, the secondheterologous polynucleotide in reverse orientation to the firstheterologous polynucleotide and a third AAV ITR.

In some embodiments, the viral particle is an adenoviral particle. Insome embodiments, the adenoviral particle is a recombinant adenoviralparticle, e.g., a polynucleotide vector comprising one or moreheterologous sequences (i.e., nucleic acid sequence not of adenoviralorigin) between two ITRs. In some embodiments, the adenoviral particlelacks or contains a defective copy of one or more E1 genes, whichrenders the adenovirus replication-defective. Adenoviruses include alinear, double-stranded DNA genome within a large (˜950 Å),non-enveloped icosahedral capsid. Adenoviruses have a large genome thatcan incorporate more than 30 kb of heterologous sequence (e.g., in placeof the E1 and/or E3 region), making them uniquely suited for use withlarger heterologous genes. They are also known to infect dividing andnon-dividing cells and do not naturally integrate into the host genome(although hybrid variants may possess this ability). In someembodiments, the adenoviral vector may be a first generation adenoviralvector with a heterologous sequence in place of E1. In some embodiments,the adenoviral vector may be a second generation adenoviral vector withadditional mutations or deletions in E2A, E2B, and/or E4. In someembodiments, the adenoviral vector may be a third generation or guttedadenoviral vector that lacks all viral coding genes, retaining only theITRs and packaging signal and requiring a helper adenovirus in trans forreplication, and packaging. Adenoviral particles have been investigatedfor use as vectors for transient transfection of mammalian cells as wellas gene therapy vectors. For further description, see, e.g., Danthinne,X. and Imperiale, M. J. (2000) Gene Ther. 7:1707-14 and Tatsis, N. andErtl, H. C. (2004) Mol. Ther. 10:616-29.

In some embodiments, the viral particle is a recombinant adenoviralparticle comprising a nucleic acid comprising a transgene. Use of anyadenovirus serotype is considered within the scope of the presentinvention. In some embodiments, the recombinant adenoviral vector is avector derived from an adenovirus serotype, including withoutlimitation, AdHu2, AdHu 3, AdHu4, AdHu5, AdHu7, AdHu11, AdHu24, AdHu26,AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6,AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, and porcineAd type 3. The adenoviral particle also comprises capsid proteins. Insome embodiments, the recombinant viral particles comprise an adenoviralparticle in combination with one or more foreign viral capsid proteins.Such combinations may be referred to as pseudotyped recombinantadenoviral particles. In some embodiments, foreign viral capsid proteinsused in pseudotyped recombinant adenoviral particles are derived from aforeign virus or from another adenovirus serotype. In some embodiments,the foreign viral capsid proteins are derived from, including withoutlimitation, reovirus type 3. Examples of vector and capsid proteincombinations used in pseudotyped adenovirus particles can be found inthe following references (Tatsis, N. et al. (2004) Mol. Ther.10(4):616-629 and Ahi, Y. et al. (2011) Curr. Gene Ther. 11(4):307-320).Different adenovirus serotypes can be used to optimize transduction ofparticular target cells or to target specific cell types within aparticular target tissue (e.g., a diseased tissue). Tissues or cellstargeted by specific adenovirus serotypes, include without limitation,lung (e.g. HuAd3), spleen and liver (e.g. HuAd37), smooth muscle,synoviocytes, dendritic cells, cardiovascular cells, tumor cell lines(e.g. HuAd11), and dendritic cells (e.g. HuAd5 pseudotyped with reovirustype 3, HuAd30, or HuAd35). For further description, see Ahi, Y. et al.(2011) Curr. Gene Ther. 11(4):307-320, Kay, M. et al. (2001) Nat. Med.7(1):33-40, and Tatsis, N. et al. (2004) Mol. Ther. 10(4):616-629.

In some embodiments, the viral particle is a lentiviral particle. Insome embodiments, the lentiviral particle is a recombinant lentiviralparticle, e.g., a polynucleotide vector comprising one or moreheterologous sequences (i.e., nucleic acid sequence not of lentiviralorigin) between two LTRs. Lentiviruses are positive-sense, ssRNAretroviruses with a genome of approximately 10 kb. Lentiviruses areknown to integrate into the genome of dividing and non-dividing cells.Lentiviral particles may be produced, for example, by transfectingmultiple plasmids (typically the lentiviral genome and the genesrequired for replication and/or packaging are separated to prevent viralreplication) into a packaging cell line, which packages the modifiedlentiviral genome into lentiviral particles. In some embodiments, alentiviral particle may refer to a first generation vector that lacksthe envelope protein. In some embodiments, a lentiviral particle mayrefer to a second generation vector that lacks all genes except thegag/pol and tat/rev regions. In some embodiments, a lentiviral particlemay refer to a third generation vector that only contains the endogenousrev, gag, and pol genes and has a chimeric LTR for transduction withoutthe tat gene (see Dull, T. et al. (1998) J. Virol. 72:8463-71). Forfurther description, see Durand, S. and Cimarelli, A. (2011) Viruses3:132-59.

In some embodiments, the viral particle is a recombinant lentiviralparticle comprising a nucleic acid comprising a transgene. Use of anylentiviral vector is considered within the scope of the presentinvention. In some embodiments, the lentiviral vector is derived from alentivirus including, without limitation, human immunodeficiency virus-1(HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiencyvirus (SIV), feline immunodeficiency virus (FIV), equine infectiousanemia virus (EIAV), bovine immunodeficiency virus (BIV), Jembranadisease virus (JDV), visna virus (VV), and caprine arthritisencephalitis virus (CAEV). The lentiviral particle also comprises capsidproteins. In some embodiments, the recombinant viral particles comprisea lentivirus vector in combination with one or more foreign viral capsidproteins. Such combinations may be referred to as pseudotypedrecombinant lentiviral particles. In some embodiments, foreign viralcapsid proteins used in pseudotyped recombinant lentiviral particles arederived from a foreign virus. In some embodiments, the foreign viralcapsid protein used in pseudotyped recombinant lentiviral particles isVesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts witha ubiquitous cell receptor, providing broad tissue tropism topseudotyped recombinant lentiviral particles. In addition, VSV-GP isthought to provide higher stability to pseudotyped recombinantlentiviral particles. In other embodiments, the foreign viral capsidproteins are derived from, including without limitation, Chandipuravirus, Rabies virus, Mokola virus, Lymphocytic choriomeningitis virus(LCMV), Ross River virus (RRV), Sindbis virus, Semliki Forest virus(SFV), Venezuelan equine encephalitis virus, Ebola virus Reston, Ebolavirus Zaire, Marburg virus, Lassa virus, Avian leukosis virus (ALV),Jaagsiekte sheep retrovirus (JSRV), Moloney Murine leukemia virus (MLV),Gibbon ape leukemia virus (GALV), Feline endogenous retrovirus (RD114),Human T-lymphotropic virus 1 (HTLV-1), Human foamy virus, Maedi-visnavirus (MVV), SARS-CoV, Sendai virus, Respiratory syncytia virus (RSV),Human parainfluenza virus type 3, Hepatitis C virus (HCV), Influenzavirus, Fowl plague virus (FPV), or Autographa californica multiplenucleopolyhedro virus (AcMNPV). Examples of vector and capsid proteincombinations used in pseudotyped Lentivirus particles can be found, forexample, in Cronin, J. et al. (2005). Curr. Gene Ther. 5(4):387-398.Different pseudotyped recombinant lentiviral particles can be used tooptimize transduction of particular target cells or to target specificcell types within a particular target tissue (e.g., a diseased tissue).For example, tissues targeted by specific pseudotyped recombinantlentiviral particles, include without limitation, liver (e.g.pseudotyped with a VSV-G, LCMV, RRV, or SeV F protein), lung (e.g.pseudotyped with an Ebola, Marburg, SeV F and HN, or JSRV protein),pancreatic islet cells (e.g. pseudotyped with an LCMV protein), centralnervous system (e.g. pseudotyped with a VSV-G, LCMV, Rabies, or Mokolaprotein), retina (e.g. pseudotyped with a VSV-G or Mokola protein),monocytes or muscle (e.g. pseudotyped with a Mokola or Ebola protein),hematopoietic system (e.g. pseudotyped with an RD114 or GALV protein),or cancer cells (e.g. pseudotyped with a GALV or LCMV protein). Forfurther description, see Cronin, J. et al. (2005). Curr. Gene Ther.5(4):387-398 and Kay, M. et al. (2001) Nat. Med. 7(1):33-40.

In some embodiments, the viral particle is a herpes simplex virus (HSV)particle. In some embodiments, the HSV particle is a rHSV particle,e.g., a polynucleotide vector comprising one or more heterologoussequences (i.e., nucleic acid sequence not of lentiviral origin) betweentwo TRs. HSV is an enveloped, double-stranded DNA virus with a genome ofapproximately 152 kb. Advantageously, approximately half of its genesare nonessential and may be deleted to accommodate heterologoussequence. HSV particles infect non-dividing cells. In addition, theynaturally establish latency in neurons, travel by retrograde transport,and can be transferred across synapses, making them advantageous fortransfection of neurons and/or gene therapy approaches involving thenervous system. In some embodiments, the HSV particle may bereplication-defective or replication-competent (e.g., competent for asingle replication cycle through inactivation of one or more lategenes). For further description, see Manservigi, R. et al. (2010) OpenVirol. J. 4:123-56.

In some embodiments, the viral particle is a rHSV particle comprising anucleic acid comprising a transgene. Use of any HSV vector is consideredwithin the scope of the present invention. In some embodiments, the HSVvector is derived from a HSV serotype, including without limitation,HSV-1 and HSV-2. The HSV particle also comprises capsid proteins. Insome embodiments, the recombinant viral particles comprise a HSV vectorin combination with one or more foreign viral capsid proteins. Suchcombinations may be referred to as pseudotyped rHSV particles. In someembodiments, foreign viral capsid proteins used in pseudotyped rHSVparticles are derived from a foreign virus or from another HSV serotype.In some embodiments, the foreign viral capsid protein used in apseudotyped rHSV particle is a Vesicular stomatitis virus glycoprotein(VSV-GP). VSV-GP interacts with a ubiquitous cell receptor, providingbroad tissue tropism to pseudotyped rHSV particles. In addition, VSV-GPis thought to provide higher stability to pseudotyped rHSV particles. Inother embodiments, the foreign viral capsid protein may be from adifferent HSV serotype. For example, an HSV-1 vector may contain one ormore HSV-2 capsid proteins. Different HSV serotypes can be used tooptimize transduction of particular target cells or to target specificcell types within a particular target tissue (e.g., a diseased tissue).Tissues or cells targeted by specific adenovirus serotypes includewithout limitation, central nervous system and neurons (e.g. HSV-1). Forfurther description, see Manservigi, R. et al. (2010) Open Virol J4:123-156, Kay, M. et al. (2001) Nat. Med. 7(1):33-40, and Meignier, B.et al. (1987) J. Infect. Dis. 155(5):921-930.

V. Production of Viral Vectors

Numerous methods are known in the art for production of rAAV vectors,including transfection, stable cell line production, and infectioushybrid virus production systems which include adenovirus-AAV hybrids,herpesvirus-AAV hybrids (Conway, J E et al., (1997) J. Virology71(11):8780-8789) and baculovirus-AAV hybrids. rAAV production culturesfor the production of rAAV virus particles all require; 1) suitable hostcells, including, for example, human-derived cell lines such as HeLa,A549, or 293 cells, or insect-derived cell lines such as SF-9, in thecase of baculovirus production systems; 2) suitable helper virusfunction, provided by wild-type or mutant adenovirus (such astemperature sensitive adenovirus), herpes virus, baculovirus, or aplasmid construct providing helper functions; 3) AAV rep and cap genesand gene products; 4) a transgene (such as a therapeutic transgene)flanked by at least one AAV ITR sequences; and 5) suitable media andmedia components to support rAAV production. In some embodiments, theAAV rep and cap gene products may be from any AAV serotype. In general,but not obligatory, the AAV rep gene product is of the same serotype asthe ITRs of the rAAV vector genome as long as the rep gene products mayfunction to replicated and package the rAAV genome. Suitable media knownin the art may be used for the production of rAAV vectors. These mediainclude, without limitation, media produced by Hyclone Laboratories andJRH including Modified Eagle Medium (MEM), Dulbecco's Modified EagleMedium (DMEM), custom formulations such as those described in U.S. Pat.No. 6,566,118, and Sf-900 II SFM media as described in U.S. Pat. No.6,723,551, each of which is incorporated herein by reference in itsentirety, particularly with respect to custom media formulations for usein production of recombinant AAV vectors. In some embodiments, the AAVhelper functions are provided by adenovirus or HSV. In some embodiments,the AAV helper functions are provided by baculovirus and the host cellis an insect cell (e.g., Spodoptera frugiperda (Sf9) cells).

Suitable rAAV production culture media of the present invention may besupplemented with serum or serum-derived recombinant proteins at a levelof 0.5%-20% (v/v or w/v). Alternatively, as is known in the art, rAAVvectors may be produced in serum-free conditions which may also bereferred to as media with no animal-derived products. One of ordinaryskill in the art may appreciate that commercial or custom media designedto support production of rAAV vectors may also be supplemented with oneor more cell culture components know in the art, including withoutlimitation glucose, vitamins, amino acids, and or growth factors, inorder to increase the titer of rAAV in production cultures.

In some aspects, the invention provides methods for preparing rAAVparticles with reduced empty capsids comprising a) culturing host cellsunder conditions suitable for rAAV production, wherein the cellscomprise i) nucleic acid encoding a heterologous transgene flanked by atleast one AAV ITR, ii) nucleic acid comprising AAV rep and cap codingregions, wherein the nucleic acid comprises a mutated p5 promoterwherein expression from the p5 promoter is reduced compared to awild-type p5 promoter, and iii) nucleic acid encoding AAV helper virusfunctions; b) lysing the host cells to release rAAV particles; c)isolating the rAAV particles produced by the host cell; and d) analyzingthe rAAV particles for the presence of empty capsids and/or rAAVparticles with variant genomes by analytical ultracentrifugation asdescribed above. In some embodiments, the p5 promoter of the nucleicacid encoding AAV rep and cap regions is located 3′ to the rep and/orcap coding region. In some embodiments, the nucleic acid encoding AAVrep and cap coding regions is plasmid pHLP, pHLP19, or pHLP09 (see U.S.Pat. Nos. 5,622,856; 6,001,650; 6,027,931; 6,365,403; 6,376,237; and7,037,713; the content of each is incorporated herein in its entirety).In some embodiments, the AAV helper virus functions comprise adenovirusE1A function, adenovirus E1B function, adenovirus E2A function,adenovirus VA function and adenovirus E4 orf6 function.

rAAV production cultures can be grown under a variety of conditions(over a wide temperature range, for varying lengths of time, and thelike) suitable to the particular host cell being utilized. As is knownin the art, rAAV production cultures include attachment-dependentcultures which can be cultured in suitable attachment-dependent vesselssuch as, for example, roller bottles, hollow fiber filters,microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vectorproduction cultures may also include suspension-adapted host cells suchas HeLa, 293, and SF-9 cells which can be cultured in a variety of waysincluding, for example, spinner flasks, stirred tank bioreactors, anddisposable systems such as the Wave bag system.

rAAV vector particles of the invention may be harvested from rAAVproduction cultures by lysis of the host cells of the production cultureor by harvest of the spent media from the production culture, providedthe cells are cultured under conditions known in the art to causerelease of rAAV particles into the media from intact cells, as describedmore fully in U.S. Pat. No. 6,566,118). Suitable methods of lysing cellsare also known in the art and include for example multiple freeze/thawcycles, sonication, microfluidization, and treatment with chemicals,such as detergents and/or proteases.

Numerous methods are known in the art for production of adenoviralvector particles. For example, for a gutted adenoviral vector, theadenoviral vector genome and a helper adenovirus genome may betransfected into a packaging cell line (e.g., a 293 cell line). In someembodiments, the helper adenovirus genome may contain recombinationsites flanking its packaging signal, and both genomes may be transfectedinto a packaging cell line that expresses a recombinase (e.g., theCre/loxP system may be used), such that the adenoviral vector ofinterest is packaged more efficiently than the helper adenovirus (see,e.g., Alba, R. et al. (2005) Gene Ther. 12 Suppl 1:S18-27). Adenoviralvectors may be harvested and purified using standard methods, such asthose described herein.

Numerous methods are known in the art for production of lentiviralvector particles. For example, for a third-generation lentiviral vector,a vector containing the lentiviral genome of interest with gag and polgenes may be co-transfected into a packaging cell line (e.g., a 293 cellline) along with a vector containing a rev gene. The lentiviral genomeof interest also contains a chimeric LTR that promotes transcription inthe absence of Tat (see Dull, T. et al. (1998) J. Virol. 72:8463-71).Lentiviral vectors may be harvested and purified using methods (e.g.,Segura M M, et al., (2013) Expert Opin Biol Ther. 13(7):987-1011)described herein.

Numerous methods are known in the art for production of HSV particles.HSV vectors may be harvested and purified using standard methods, suchas those described herein. For example, for a replication-defective HSVvector, an HSV genome of interest that lacks all of the immediate early(IE) genes may be transfected into a complementing cell line thatprovides genes required for virus production, such as ICP4, ICP27, andICP0 (see, e.g., Samaniego, L. A. et al. (1998) J. Virol. 72:3307-20).HSV vectors may be harvested and purified using methods described (e.g.,Goins, W F et al., (2014) Herpes Simplex Virus Methods in MolecularBiology 1144:63-79).

VI. Purification of rAAV Vectors

At harvest, rAAV production cultures of the present invention maycontain one or more of the following: (1) host cell proteins; (2) hostcell DNA; (3) plasmid DNA; (4) helper virus; (5) helper virus proteins;(6) helper virus DNA; and (7) media components including, for example,serum proteins, amino acids, transferrins and other low molecular weightproteins. In addition, rAAV production cultures further include rAAVparticles having an AAV capsid serotype selected from the groupconsisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8,AAV9, AAV10, AAVrh10, AAV11, or AAV12. In some embodiments, the rAAVproduction cultures further comprise empty AAV capsids (e.g., a rAAVparticle comprising capsid proteins but no rAAV genome). In someembodiments, the rAAV production cultures further comprise rAAVparticles comprising variant rAAV genomes (e.g., a rAAV particlecomprising a rAAV genome that differs from an intact full-length rAAVgenome). In some embodiments, the rAAV production cultures furthercomprise rAAV particles comprising truncated rAAV genomes. In someembodiments, the rAAV production cultures further comprise rAAVparticles comprising AAV-encapsidated DNA impurities.

In some embodiments, the rAAV production culture harvest is clarified toremove host cell debris. In some embodiments, the production cultureharvest is clarified by filtration through a series of depth filtersincluding, for example, a grade DOHC Millipore Millistak+HC Pod Filter,a grade A1HC Millipore Millistak+HC Pod Filter, and a 0.2 μm FilterOpticap XL10 Millipore Express SHC Hydrophilic Membrane filter.Clarification can also be achieved by a variety of other standardtechniques known in the art, such as, centrifugation or filtrationthrough any cellulose acetate filter of 0.2 μm or greater pore sizeknown in the art.

In some embodiments, the rAAV production culture harvest is furthertreated with Benzonase® to digest any high molecular weight DNA presentin the production culture. In some embodiments, the Benzonase® digestionis performed under standard conditions known in the art including, forexample, a final concentration of 1-2.5 units/ml of Benzonase® at atemperature ranging from ambient to 37° C. for a period of 30 minutes toseveral hours.

rAAV particles may be isolated or purified using one or more of thefollowing purification steps: equilibrium centrifugation; flow-throughanionic exchange filtration; tangential flow filtration (TFF) forconcentrating the rAAV particles; rAAV capture by apatitechromatography; heat inactivation of helper virus; rAAV capture byhydrophobic interaction chromatography; buffer exchange by sizeexclusion chromatography (SEC); nanofiltration; and rAAV capture byanionic exchange chromatography, cationic exchange chromatography, oraffinity chromatography. These steps may be used alone, in variouscombinations, or in different orders. In some embodiments, the methodcomprises all the steps in the order as described below. Methods topurify rAAV particles are found, for example, in Xiao et al., (1998)Journal of Virology 72:2224-2232; U.S. Pat. Nos. 6,989,264 and 8,137,948and WO 2010/148143. Methods to purify adenovirus particles are found,for example, in Bo, H et al., (2014) Eur. J. Pharm. Sci. 67C:119-125.Methods to purify lentivirus particles are found, for example, in SeguraM M, et al., (2013) Expert Opin Biol Ther. 13(7):987-1011. Methods topurify HSV particles are found, for example, in Goins, W F et al.,(2014) Herpes Simplex Virus Methods in Molecular Biology 1144:63-79.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1: Characterization of Recombinant Adeno-Associated Viral VectorPreparations by Analytical Ultracentrifugation

Adeno-associated viruses (AAV) have features that make them attractiveas vectors for gene therapy. Wild-type AAV consists of two open readingframes (rep and cap) which code for all structural and regulatoryelements required for assembly, replication and infection. The rep ORFcodes for Rep 78 and 68 proteins which have genome replication functionsas well as Rep 52 and 40 proteins which are involved in single strandreplication and packaging. The cap ORF codes for the three structuralcapsid proteins: VP1, VP2 and VP3. Recombinant AAV vector is typicallyproduced by the triple transfection method using the “gutless” vectorapproach (Xiao, X, et al., 1998, J. Virol. 3:2224-2232). The rep and capgenes are replaced with the therapeutic gene and its regulatory elementssandwiched between a 5′ and 3′ inverted terminal repeat (ITR), the repand cap genes are provided in trans on a separate plasmid and a thirdplasmid contributes required adenoviral helper genes. It is postulatedthat the viral capsids are fully assembled and the ITR flanked vectorgenome is then inserted into the capsid via a capsid pore (Myers, M W &Carter, B J, 1980, Virology, 102:71-82). The resulting population ofcapsids contains both non-genome containing capsids (empty capsids) aswell as genome containing capsids. In addition, capsids may containincomplete portions of the recombinant viral genome. The vector prep maythen purified by affinity chromatography to isolate the capsids from thecellular debris and can be further processed to enrich for intact vectorby anion exchange chromatography.

Based on their recent approval for use in gene therapy, adeno-associatedviral (AAV) vectors have emerged as an important class of novelbiopharmaceutical drug products. The generation of AAV vector productsrequires an analytical method that monitors product quality with regardto homogeneity, purity, and consistency of manufacturing, yet to date nomethod to support AAV vector characterization has been established. Tomeet this demand, the potential use of analytical ultracentrifugation(AUC) as a technique to characterize the homogeneity of AAV vectors wasinvestigated.

Methods

Sample Preparation

In order to support accurate AUC assessment, vector product(AAV2-transgene 2) was highly purified, suitably buffered, andconcentrated to greater than 5×10¹¹ vg/mL. To achieve this, cellsupernatants runs were purified using AVB affinity chromatography (GEHealthcare) and buffer-exchanged into PBS, pH 7.2 using a 10K MWCOSlide-a-Lyzer (Thermo Scientific). Product concentration was determinedby optical density measurement at 260 nm (OD₂₆₀) by spectrophotometricmethods. To generate reproducible and consistent AUC data, sampleadjustments were made to target concentration by optical densitymeasurement at 260 nm from 0.1 to 1.0, either by direct dilution withPBS or further concentration using Amicon Ultra-0.5/30K MWCO CentrifugalFilter Device.

Sedimentation Velocity AUC Data Acquisition

Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysiswas performed using a ProteomeLab™ XL-I (Beckman Coulter). 400 μL samplewas loaded into the sample sector of a two sector velocity cell, and 400μL PBS was loaded into the corresponding reference sector. The samplewas placed in the four-hole rotor and allowed to equilibrate in theinstrument until a temperature of 20° C. and full vacuum were maintainedfor one hour. Sedimentation velocity centrifugation was performed at20,000 RPM, 20° C., 0.003 cm radial step setting, with no delay and withno replicates. Absorbance (260 nm) and Raleigh interference optics wereused to simultaneously record radial concentration as a function of timeuntil the smallest sedimenting component cleared the optical window (1.2hour). Assay throughput was limited to a single sample per run based onabsorbance scan collection times of greater than one minute, as well asthe large size and rapid sedimentation of AAV.

AUC Data Analysis

The percent full capsid was determined by analyzing approximately 75scans from each detection method using the SEDFIT (NIH/see worldwide webat analyticalultracentrifugation.com) continuous size C(S) distributionmodel. Second (2^(nd)) derivative regularization was applied to thefitting with a confidence level of F statistic=0.68. The following C(S)parameters were held constant: resolution=2005, S min=1, S max=200 andfrictional ratio=1.0. RI and TI noise subtractions were applied, and themeniscus position was allowed to float, letting the software choose theoptimal position. This model fit the data to the Lamm equation, and theresulting size distribution was a “distribution of sedimentationcoefficients” that looked like a chromatogram with the area under eachpeak proportional to concentration in units of Fringes or OD₂₆₀ units.The sedimentation coefficient (in Svedberg units) and the relativeconcentration (in OD units) were determined for each component in thedistribution. Each AUC run was an independent assay, and each analysiswas monitored for the following attributes to ensure quality of results:goodness of fit (rmsd), the ratio of OD_(260 nm)/interference signal infringes (A260/IF ratio) for each peak, consistency of sedimentationcoefficients for each species between runs, and overall quality of thescans.

Absorbance Optics (260 nm)

Extinction coefficients were used to calculate molar concentration andthe actual percent value of the intact vector peak from absorbance data.Molar absorbance extinction coefficients for both empty capsids(€_(260/capsid)=3.72e6) and intact vector (€_(260/vector)=3.00e7) werecalculated based on published formulae (Sommer et al. (2003) Mol Ther.,7:122-8). Extinction coefficients were available for empty capsid andintact vector peaks. The C(S) values were determined using the SEDFITalgorithm described by Schuck (2000) Biophys. J., 78:1606-19. Molarconcentration of both intact vector and empty capsid were calculatedusing Beer's Law, and the percentage of full capsid was calculated fromthese values. Values were reported in terms of the percentage of fullcapsid.

Generation of an AUC Standard Curve

Because it is not possible to determine empirically the extinctioncoefficient of fragmented genomes of unknown size and sequence, arelationship between S value and genome size was established. To achievethis, rAAV vector preps with encapsidated viral genomes of knownnucleotide size were analyzed by AUC, and a corresponding S value wasdetermined as described above.

Production of rAAV by Transient Transfection

Recombinant AAV vector was produced by the triple transfection methodusing the “gutless” vector approach (Xiao et al. (1998) J. Virol.,3:2224-32). In this approach, the rep and cap genes were replaced withthe therapeutic gene and its regulatory elements, both sandwichedbetween a 5′ and 3′ inverted terminal repeat (ITR). The rep and capgenes were provided in trans on a separate plasmid, and a third plasmidcontributed the required adenoviral helper genes. Without wishing to bebound to theory, it is postulated that the viral capsids are fullyassembled, and the ITR flanked vector genome is then inserted into thecapsid via a capsid pore (Myers & Carter (1980) Virology, 102:71-82).The resulting population of capsids contained both non-genome-containingcapsids (empty capsids) and genome-containing capsids.

Production of rAAV by Producer Cell Platform

The AAV producer cell line is an alternative production platform used togenerate clinical rAAV vectors. With this method, a HeLa S3 cell,adapted to growth in suspension, was engineered to have integratedcopies of the AAV rep and cap genes required for vector replication andpackaging, in addition to vector sequences and the selectable marker(see, e.g., Puro: Thorne et al. (2009) Hum. Gene Ther., 20:707-14). Onceinfected with WT Adenovirus, which provides the helper functionsrequired for replication, the cell produced recombinant AAV vector aswell as adenovirus, which was subsequently removed during thepurification process using ion-exchange chromatography.

Other Methods

Synthetic transgenes were cloned into a plasmid that contained apromoter of choice and bovine growth hormone polyadenylation signalsequence (polyA). The entire transgene expression cassette was thencloned into previral plasmid vector pAAVDC64 containing AAV2 invertedterminal repeats. The total size of the resulting AAV genomes in therespective expression plasmids (including the region flanked by ITRs)was 4-4.6 kb. The recombinant vectors were produced by tripletransfection of 293 cells using helper plasmids expressing rep2/capsequences and Adenovirus helper functions, pAd Helper (Stratagene, LaJolla, Calif. USA) The rep/cap helper expressed rep from AAV serotype 2,while the cap sequence encoded one of the following sequences: AAV cap1, 2, 5, 9, or rh8R. Vectors were purified by affinity chromatographyand in some cases were further purified to remove empty particles (see,e.g., Qu et al. (2007) J. Virol. Methods. 140:183-92).

Results

Analytical ultracentrifugation (AUC) using classical boundarysedimentation velocity was used to reveal the particle heterogeneitiesof recombinant adeno-associated virus (rAAV) vector preps. A mixturecontaining 20% rAAV2 particles with the full genome and 80% emptycapsids was created by mixing together purified empty capsids andpurified genome-containing capsids at defined ratios. The empty and fullcapsids were generated by CsCl₂ gradient purification of a mixture ofempty and full capsids following triple transfection production. Tomonitor the movement of rAAV2 particles in response to a centrifugalforce, this mixture of rAAV2 capsids was scanned at an absorbance of 260nm along a centrifugal field at defined time intervals. FIG. 1A shows arepresentative scanning profile following centrifugation of the AAV2mixture at 20,000 rpm for 1.2 hrs (until the smallest sedimentingspecies cleared the optical window). Scans represented the acquisitionof concentration data as a function of radius r, at times t, to yield aseries of concentration scans that revealed the complete migrationpattern of constituent vector particles in the rAAV2 vector prep. Inthese sigmoidal curves or boundaries, the leading edge of the curverepresented the faster sedimenting species (i.e., the genome-containingrAAV2 capsid), and the trailing edge of the curve represented the slowersedimenting species (i.e., the “empty” rAAV2 capsids) (FIG. 1A).

Plotting the differential sedimentation coefficient distribution value,C(S), versus the sedimentation coefficient (in Svedberg units, S)yielded distinct peaks with unique sedimentation coefficients for boththe empty and genome-containing capsid species (FIG. 1B). The C(S)values were determined using the SEDFIT algorithm described by Schuck(2000) Biophys. J., 78:1606-19. In order to calculate molarconcentrations and percent value for each capsid species from theabsorbance data, extinction coefficients were used according to Table 2.

TABLE 2 Extinction coefficients and molar concentrations for capsidspecies Signal Molar concentration Relative Species (Abs_(260 nm))ε_(260 nm) (M) abundance (%) Peak 1 0.0479 3.73E+06 1.28E−08 79 Peak 20.0909 2.59E+07 3.51E−09 21 Sum 1.63E−08

Molar absorbance extinction coefficients for both the empty capsid(ε_(260/capsid)=3.72e6) and genome containing capsid(ε_(260/capsid)=3e7) were calculated using genome size and publishedformulae (Sommer et al., 2003). Molar concentrations of bothgenome-containing and empty capsids were then calculated using Beer'sLaw. The molar concentration of each species was used to calculate itsrelative abundance, expressed as a percentage of total capsids (FIG. 1).These results demonstrated that AUC may be used to accuratelydistinguish and quantify empty capsids and genome-containing capsidsfrom a heterogeneous vector preparation.

For the rAAV2 vector prep shown in FIG. 1, capsids containing a fullgenome were represented by the peak sedimenting at 94 S and accountedfor 21% of the vector prep. Empty capsids sedimented with an S value of64 S and accounted for 79% of the vector prep. These sedimentationcoefficient values were confirmed by AUC analysis of pure populations ofempty (FIG. 2A) or genome containing particles (FIG. 2B). The AUCprofile of a pure population of rAAV2 empty capsids revealed a singlepeak with a sedimentation coefficient of 64 S, whereas the AUC profileof a pure population of rAAV2AUC genome-containing capsids revealed asingle peak with a higher sedimentation coefficient of 94 S. Theseresults agreed with the values generated from a heterogeneouspreparation and further confirmed that AUC methods may be used toquantify genome-containing and empty AAV capsids from a heterogeneouspreparation containing both species.

The AUC method was further assessed for reproducibility by performingfive independent AUC runs of the same vector sample (scAAV2/9 LP2), asshown in Table 3. The sedimentation coefficients for bothgenome-containing and empty AAV2 capsids were highly reproducible,yielding coefficients of variation from 0.5-0.6%. Similarly, therelative abundance (expressed as a percentage of the total) ofgenome-containing capsids was determined with a coefficient of variationof approximately 2%. These results indicated that the AUC method forquantifying genome-containing and empty AAV2 capsids yields highlyreproducible and consistent values.

TABLE 3 Five independent assays on scAAV2/9 LP2 sample. Empty Capsid,Peak 1 Full Capsid, Peak 3 AUC Run (S) % Full Capsids S 20110927A 64.334.1 84.0 20111003A 64.6 34.2 84.6 20111005A 65.1 35.0 84.5 20111005B64.2 35.2 83.8 20111011A 64.1 33.5 84.5 Mean 64.5 34.4 84.3 StandardDeviation 0.4 0.7 0.4 % CV 0.6 2.1 0.5

Example 2: Comparison of Interference and Absorbance Detection Methodsfor AUC

An alternative optical detection method for AUC, Rayleigh InterferenceOptics, was also evaluated. This detection method measures the sampleconcentration based on refractive index differences between a referencesolution and the AAV containing sample. Like absorbance detection,interference detection can be applied to any rAAV regardless of thesequence of the genome. Unlike absorbance detection, which requires anextinction coefficient, interference detection yields integrated peaksthat are directly proportional to concentration.

Pure populations of empty and genome-containing AAV2 capsids were mixedat a 1:1 ratio and analyzed by AUC using both interference (FIG. 3A) andabsorbance detection methods (FIG. 3B). Interference detection revealedtwo populations of AAV capsids at the approximate expected ratios of 43%empty and 57% genome-containing (FIG. 3A). Both detection methodsyielded similar abundance ratios. However, comparing the peak sizesgenerated by both methods illustrates the disconnect between peak heightand concentration with absorbance detection (compare size of “emptycapsid” peaks in FIGS. 3A and 3B). The data generated by both methodsare compared in Table 4. The ratio of absorbance signal to interferencesignal (A_(260 nm)/IF) can be used in a fashion analogous to the 260/280ratio of absorbance data, and this assisted in identifying peaks in theC(S) distribution.

TABLE 4 S values and relative abundance generated by absorbance andinterference detection. Absorbance (260 nm) Interference RelativeRelative Sedimentation abundance Sedimentation Signal abundance Peakcoefficient (S) (%) coefficient (S) (fringes) (%) A_(260 nm)/IF Peak 163 47 62 0.080 43 0.41 Peak 2 93 53 92 0.104 57 2.38 Sum 0.184

Although interference optics offers precision and resolution, it mayrequire a high concentration of sample. Moreover, interference opticsmay be affected by a mismatch between the reference and AAV samplebuffer. AAV samples, however, typically contain a low proteinconcentration, and it may be necessary to completely match the AAVsample and reference buffers.

Example 3: Influence of Production Method on AAV Vector Heterogeneity

The previous examples demonstrated that the AUC method is a highlyaccurate and reproducible way to resolve and quantify empty andgenome-containing AAV capsids from a heterogeneous mixture. Thiscapability could be advantageous for a variety of applications toevaluate the quality of AAV vector preparations. For example, a majorproblem in producing pure AAV vector preparations is the presence ofcapsids with partial or fragmented genomes. To illustrate the utility ofthe AUC method for resolving these species, AAV vectors generated by twodifferent methods, termed the “triple transfection” and “producer cellline” methods, were analyzed by AUC.

An AAV2 vector harboring the transgene 2 was produced using either thetriple transfection method (FIG. 4) or the producer cell line method(FIG. 5). For a description of these methods, see Example 1. Followingchromatographic purification, both vector preps were analyzed by AUC.FIG. 6A shows a schematic of this AAV2 vector genome.

The AUC profiles of vector preps produced by these methods wereremarkably different. Using the producer cell line method, 74% ofcapsids contained a full genome, represented by the 92 S species (FIG.6B). 19% were empty capsids, with the remainder containing a fragmentedgenome (75 S species, 7%). In contrast, 82% of the capsids produced bythe triple transfection method were empty, 64 S species (FIG. 6C), with11% of capsids containing a fragmented genome (76 S and 84 S species)and only 8% of capsids having a full genome (94 S).

These results demonstrate that vector preparations generated using theproducer cell line technology may have high quality, containingpredominantly capsids with a full genome. The vast majority of capsidsproduced by the triple transfection method are empty, with a greaterproportion of capsids having a fragmented genome. These results alsohighlight the ability of the AUC method to resolve capsids withfragmented genomes, in addition to full genome-containing and emptycapsids. Moreover, they illustrate the power of the AUC method inevaluating the quality and homogeneity of vector preparations.

Example 4: Use of AUC to Assess Removal of Empty Capsids from VectorPreps

The AUC method was evaluated as a tool to monitor removal of emptycapsids using chromatographic methods (for methods, see Qu et al. (2007)J. Virol. Methods, 140:183-92). Separation of empty andgenome-containing capsids was performed using anion exchangechromatography (FIG. 7A). AUC was performed on the resolved peaks todemonstrate that the genome-containing rAAV2 particles were enriched inthe later fractions eluted from the resin (“Full Genome Capsid” in FIG.7A).

As shown in FIG. 7B, AUC analysis revealed that genome-containingcapsids represented 94% of the vector prep upon elution from the column.This later fraction yielded a single peak with a sedimentationcoefficient of 92 S. In contrast, the rAAV2 vector prep prior to thechromatographic step (FIG. 7C) had a substantial level of empty capsids.AUC analysis revealed two peaks with S values of 63 and 93, with the 63S peak (empty capsid) representing 52% of the total capsid population.These results show that chromatographic methods are highly effective inremoving empty capsids from AAV vector preparations Importantly, theydemonstrate the utility of applying the AUC method to evaluate vectorquality upon purification. The AUC method is a useful tool forevaluating different vector purification protocols or techniques.

Example 5: Assessment of Viral Genome Integrity by the AUC Method

As illustrated in Example 3, AAV vector preparations may contain capsidspackaged with fragmented genomes, in addition to full genome and emptyspecies. A major problem in generating homogeneous AAV preparations fortherapeutic or research applications is the presence of capsids withfragmented genomes, which may result in aberrant or absent expression oftransgenes of interest. Indeed, heterogeneity associated with AAV vectorpreps has been reported to result from packaging of fragmented genomesor AAV-encapsidated DNA impurities. (Kapranov et al. (2012) Hum. GeneTher., 23:46-55). Therefore, AUC was evaluated as a tool to quantify theaberrant packaging of fragmented genomes in rAAV vector preps.

Because it is not possible to determine empirically the extinctioncoefficient of fragmented genomes of unknown size and sequence, arelationship between S value and genome size was established. To achievethis, rAAV vector preps with encapsidated viral genomes of known sizewere analyzed by AUC, and their corresponding S values were determined,as shown in Table 5. A standard curve was then generated to correlategenome size and S value (FIG. 8). This demonstrated a highly linearrelationship (R²=0.9978) between sedimentation coefficient and genomesize.

TABLE 5 S values for rAAV vectors with known genome size. PredictedTrend Line Sedimentation Calculated values coefficient (S) Genome size(# NT) Extinction coefficient (y) (x) MW (260 nm) Empty capsid N/A3.72E+06 74  880 2.7E+05 9.17E+06 78 1421 4.4E+05 1.25E+07 82 19616.1E+05 1.59E+07 84 2232 6.9E+05 1.75E+07 88 2772 8.6E+05 2.09E+07 923313 1.0E+06 2.42E+07 96 3853 1.2E+06 2.76E+07 100  4394 1.4E+063.09E+07 104  4934 1.5E+06 3.43E+07 108  5475 1.7E+06 3.76E+07

To demonstrate the utility of AUC to detect genome fragments, aself-complementary vector comprising AAV2 ITRS, a minimal CBA promoter,and an EGFP transgene was packaged into an AAV9 capsid(AAV2/9minCBAEGFP; see schematic in FIG. 9A). The vector particles werepurified to eliminate empty capsids and analyzed by AUC. The standardcurve was then used to assign genome size to each of the resolved genomecontaining capsids. Approximately 25% of the vector prep sedimented as a1015 species, representing an encapsidated genome of ˜4.3 kb (FIG. 9A).This 1015 peak represented the double stranded dimeric vector genome,which has a predicted size of ˜4.3 kb. However, the majority of thevector prep (75%), sedimented with an S value of 82, which correspondsto a vector genome size of ˜2 kb (FIG. 9A), consistent with packaging ofthe single stranded monomer. The packaging of monomeric genomes withself-complementary vectors is well documented and is often a result ofinadvertent terminal resolution at spurious “trs like” sequences despitethe presence of an ITR with a mutated D sequence (McCarty et al. (2001)Gene Ther., 8:1248-54).

FIG. 9B shows an alkaline Southern blot of the same vector, scAAV9 EGFP,which revealed two vector populations with genomes ˜4.3 kb and ˜2 kb insize, corroborating the AUC data in FIG. 9A. The Southern blot alsoconfirmed that the monomeric viral genome was preferentially packagedover the dimeric genome. Interestingly, AUC analysis of a singlestranded AAV9 EGFP vector (˜4 kb) revealed a single predominant peakwith a measured S value of 99 S, corresponding to approximately 4.1 kbby the standard curve and 84% of capsid abundance (FIG. 9C). Theseresults suggest that single stranded AAV vectors may be packaged in amore homogeneous manner than double stranded vectors. Again, inagreement with the AUC method, Southern blot analysis of this vectorprep revealed homogeneous encapsidation of a viral genome of thepredicted size of ˜4 kb (lane 2, FIG. 9B). These results demonstratethat the AUC method may be used to measure the size of AAV vectorgenomes, yielding genome size data in agreement with the standardSouthern blotting technique. Using the AUC method, single stranded AAVvectors were found to produce more homogeneous vector preparations thandouble stranded ones. These results show that the AUC method is apowerful tool to identify and quantify capsid species with incompletegenomes from vector preparations.

Example 6: Use of AUC to Assess Factors that Influence Packaging ofVector Genomes

The AUC method was next used as a tool to identify factors thatinfluence the packaging of intact AAV vector genomes.

As discussed in Example 3, the production of rAAV vectors by transienttransfection methods requires the use of three plasmids including arep/cap helper, an ITR vector plasmid, and a pAd helper (see FIG. 4).AUC was used to assess the effect of the rep/cap helper on vector genomepackaging for both single stranded and self-complementary AAV vectors.First, a self-complementary AAV vector harboring an EGFP transgene (FIG.10A) was produced using one of two methods. In the first method (FIG.10B), a helper plasmid was used in which rep 78/68 expression was drivenby the endogenous p5 promoter (“WT Rep” construct). In the second method(FIG. 10C), the helper was modified such that 78/68 expression wasreduced by moving the p5 promoter downstream of the cap2 sequence aswell as mutating the TATA box (“pHLP Rep” construct). The full scAAV2EFGP capsid was predicted to have a sedimentation coefficient of 100 Sin dimeric genome form and 80 S in monomeric genome form (FIG. 10A).

AUC analysis of these scAAV2EGFP vector preps revealed a significantdifference in vector genome packaging. In the presence of reducedrep78/68 (pHLP), more than half (55%) of the vector prep containeddimeric genomes, represented by the 100 S species (FIG. 10C). This wasthe expected sedimentation coefficient for a capsid containing a dimericgenome of 4.4 kb. In contrast, the scAAV2EGFP prep generated with thefull complement of rep78/68 had significantly less packaged dimericgenomes (26%), with the majority of the capsids containing monomericgenomes and sedimenting at 80 S (FIG. 10B). These results uncovered asignificant difference in genome packaging induced by shifting the P5promoter of the helper plasmid, leading to reduced rep 78/68 proteinlevels.

A single stranded AAV5 Factor IX vector, AAV5FIX, (FIG. 11A-B) and asingle stranded AAV5hSMN vector (FIG. 11C-D) were generated usingrep/cap helpers that differed in rep expression as described above, butcap sequences of AAV2 were replaced by cap sequences of AAV5. Based onthe nucleotide size of the FIX expression cassette (4.3 kb), thepredicted sedimentation coefficient for the AAV5 FIX vector capsid wasapproximately 1015. AUC analysis of AAV2/5FIX made in the presence ofreduced rep78/68 (“pHLP19 Rep”) revealed a homogenous profile with themajority of the vector (90%) sedimenting with an S value of the expectedsize, ˜101 S (FIG. 11A). In contrast, AAV5 FIX vector generated using arep/cap5 helper expressing wild-type levels of rep 78/68 proteins (“WTRep”) generated a strikingly different AUC profile (FIG. 11B). Insteadof a predominant peak at 1015, this profile revealed more capsidheterogeneity, with the majority of the AAV5 FIX (80%) sedimenting at alower S value of 86 S, likely representing packaging of a fragmentedgenome. Moreover, in this vector sample only 15% of the AAV5 FIX vectorcapsids sedimented at the correct S value of −104 S (FIG. 11B).

AAV5SMN vectors made using these same wild-type and mutated p5 rep/caphelpers also had strikingly different AUC profiles. As seen with thesingle stranded AAV5FIX vectors, AAV5 SMN vectors generated in thepresence of reduced rep78/68 showed less heterogeneity by AUC analysis,with a single capsid species sedimenting at the S value of 1015,consistent with packaging of a genome of the predicted size of ˜4.4 kb(FIG. 11C). In contrast, the AUC profile for the same vector genomepackaged using “wild-type” levels of rep78/68 protein revealed threedistinct AAV vector species, with sedimentation coefficients of 100 S(predicted S value for the full vector genome of 4400 nt), 92 S(representing a fragmented genome of approximately 3300 nt) and 80 S(representing a fragmented genome of 2000 nucleotides) (FIG. 11D). Theseresults confirm a significant difference in genome packaging induced byshifting the P5 promoter of the helper plasmid using two additional AAVvectors.

Further analysis of the AAV5SMN and AAV5FIX vector preps was performedby Southern blot analysis of vector DNA. In agreement with the AUCmethod, Southern blot analysis of AAV5SMN generated with wild-typerep78/68 protein levels revealed packaging of full length (4.4 kb) andfragmented (less than 4.4 kb) SMN genomes (FIG. 12A, lane 1). Incontrast, the AAV5SMN vector generated in the presence of reducedrep78/68 protein contained largely capsids with a full length SMN genome(FIG. 12A, lane 2). Interestingly, a comparison of the two AAV5FIXvector preps by Southern analysis revealed the presence of a FIX fulllength genome even when vector was produced in the presence of wild-typelevels of rep 78/68 (FIG. 12B, lane 2). However, AUC analysis of thisAAV5FIX vector (FIG. 11B) showed that 80% of the capsids contained afragmented genome (˜3000 nucleotides), which were undetected by the FIXprobe.

Further analysis of the FIX vector preps generated under the twoexperimental conditions was performed by generating probes to discreteregions of the vector plasmid, including regions of the backbone. A mapof this vector is provided in FIG. 13. FIG. 14 shows Southern blottinganalyses using these probes to compare these FIX vector preps generatedunder different conditions. As shown in FIG. 14A, both vectorpreparations (pHLP rep, lane 1; WT rep, lane 2) contained the hFIXtransgene. However, FIG. 14B lane 2 confirms that the vector genomespecies sedimenting at 86 S observed in the WT Rep preparation (FIG.11B) was a ˜3 kb fragment. Moreover, this species reacted with anAmp^(R) specific probe (FIG. 14B, lane 2), suggesting that packagingupstream of the 5′ITR had occurred in a rep dependent manner. Incontrast, there was no evidence of an Amp^(R) containing fragment in therAAV5 FIX vector preps that were generated in the presence of reducedlevels of rep 68/78 (FIG. 14B, lane 1).

DNA impurities in the AAV FIX preps were also assessed by Q-PCR usingprimers and probes specific for Amp^(R). By Q-PCR, approximately 35%Amp^(R) titer was detected in AAV5FIX vector preps generated in thepresence of “wt” rep, in contrast to less than 1% when the same vectorplasmid was used to generate AAV5FIX vector in the presence of reducedrep68/78 (data not shown). These results underscore the utility of AUCanalysis for revealing the presence of packaged genomes that wouldotherwise go undetected by gene specific Southern blot analysis.

The packaging capacity of AAV vectors has been studied extensively, andalthough numerous reports have demonstrated successful transduction withvectors packaging oversized AAV genomes, the latter have been shown tobe fragmented into subgenomic—length DNA. To further explore theapplicability of the AUC method, the heterogeneity of AAV vectorsproduced using oversized genomes was evaluated. An expression cassetteharboring the full length CBA promoter driving expression of theβ-phosphodiesterase transgene was packaged as an oversized genome of 5.4kb (FIG. 15A) or as a wild type size genome of 4.6 kb (FIG. 15B). Togenerate the 4.6 kb genome, the CBA promoter was truncated by reducingthe size of the intron as previously reported (Gray, S J et al., (2011)Hum. Gene Ther. 22(9):1143-1153).

As shown in FIG. 15A, the AUC profile of the AAV vector prep generatedusing the oversized vector genome demonstrated that nearly half of thevector prep sedimented as a 93 S species, consistent with packaging afragmented vector genome of approximately 3.5 kb. 30% of the preparationwas represented by another sub-genomic vector species of approximately4.9 kb sedimenting at 1055. There was no evidence of packaging of afull-length 5.4 kb genome, which was predicted to sediment at 108-109 S.In contrast, AUC analysis revealed that the same transgene under controlof the abbreviated CBA promoter sedimented predominantly as a vectorspecies of 102 S, consistent with packaging of the predicted,full-length vector genome of 4.6 kb (FIG. 15B). These resultsdemonstrate the utility of AUC analysis in profiling AAV vectors withoversized genomes, and this profiling is critical, given the observedincidence of genome fragmentation.

This example demonstrated that the AUC method is highly effective inanalyzing the genome size of AAV vector capsids in a heterogeneouspreparation. By resolving genome-containing capsids by size (e.g.,dimeric and monomeric genomes, or partial fragments thereof), the AUCmethod represents a powerful tool for assaying the quality of AAV vectorpreps produced under different conditions. Moreover, the results fromthree distinct vector systems demonstrated that the AUC method is widelyuseful for quality control and optimization of conditions to yieldimproved AAV vector preparations Importantly, the AUC method is able todetect fragmented genomes that are not detectable by Southern blotanalysis. Whereas Southern blotting relies on the presence of DNA probesequence for detection, the AUC method is sequence-independent. The AUCmethod has also been demonstrated to be an effective tool in analyzingoversized AAV genomes. In total, these results demonstrate the highlyadvantageous and effective implementation of AUC methods to analyzemultiple types of AAV vector preparations, which have been found todisplay dramatically variable effects on genome packaging.

Example 7: Characterization of Recombinant Adenoviral VectorPreparations by Analytical Ultracentrifugation

Adenovirus (Ad) vectors have features that make them attractive asvectors for gene therapy. The generation of Ad vector products requiresan analytical method that monitors product quality with regard tohomogeneity, purity, and consistency of manufacturing. To meet thisdemand, the potential use of analytical ultracentrifugation (AUC) as atechnique to characterize the homogeneity of Ad vectors wasinvestigated.

Methods

Sample Preparation

In order to support accurate AUC assessment, a recombinant adenovirusserotype 2 vector (Ad2) was prepared and highly purified ty CsClgradient ultrafiltration to enrich for genome containing particles.Product concentration was determined by optical density measurement at260 nm (OD₂₆₀) by spectrophotometric methods. To generate reproducibleand consistent AUC data, sample adjustments were made to targetconcentration by optical density measurement at 260 nm from 0.1 to 1.0,either by direct dilution with PBS or further concentration using AmiconUltra-0.5/30K MWCO Centrifugal Filter Device.

Sedimentation Velocity AUC Data Acquisition

Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysiswas performed using a ProteomeLab™ XL-I (Beckman Coulter). 400 μL samplewas loaded into the sample sector of a two sector velocity cell, and 400μL PBS was loaded into the corresponding reference sector. The samplewas placed in the four-hole rotor and allowed to equilibrate in theinstrument until a temperature of 20° C. and full vacuum were maintainedfor one hour. Sedimentation velocity centrifugation was performed at6,000 RPM, 20° C., 0.003 cm radial step setting, with no delay and withno replicates. Raleigh interference optics were used to simultaneouslyrecord radial concentration as a function of time until the smallestsedimenting component cleared the optical window (1.2 hour). Assaythroughput was limited to a single sample per run based on absorbancescan collection times of greater than one minute, as well as the largesize and rapid sedimentation of Ad2.

AUC Data Analysis

The percent full capsid was determined by analyzing approximately 75scans from interference detection method using the SEDFIT (NIH/seeworldwide web at analyticalultracentrifugation.com) continuous size C(S)distribution model. Second (2^(nd)) derivative regularization wasapplied to the fitting with a confidence level of Fstatistic/ratio=0.68. The following C(S) parameters were held constant:resolution=2505, S min=10, S max=1500 and frictional ratio=1.86935. RIand TI noise subtractions were applied, and the meniscus position wasallowed to float, letting the software choose the optimal position. Thismodel fit the data to the Lamm equation, and the resulting sizedistribution was a “distribution of sedimentation coefficients” thatlooked like a chromatogram with the area under each peak proportional toconcentration in units of Fringes or OD₂₆₀ units. The sedimentationcoefficient (in Svedberg units) and the relative concentration (in ODunits) were determined for each component in the distribution. Each AUCrun was an independent assay, and each analysis was monitored for thefollowing attributes to ensure quality of results: goodness of fit(rmsd), the ratio of OD_(260 nm)/interference signal in fringes (A260/IFratio) for each peak, consistency of sedimentation coefficients for eachspecies between runs, and overall quality of the scans. The rmsd of thisrepresentative example was 0.006584.

Results

Analytical ultracentrifugation (AUC) using classical boundarysedimentation velocity was used to reveal the particle heterogeneitiesof recombinant adenovirus serotype 2 vectdor (rAd2) vector preps. Tomonitor the movement of rAd2 particles in response to a centrifugalforce, this mixture of rAd2 capsids was scanned using interferenceoptics along a centrifugal field at defined time intervals. Scansrepresented the acquisition of concentration data as a function ofradius r, at times t, to yield a series of concentration scans thatrevealed the complete migration pattern of constituent vector particlesin the rAd2 vector prep. Plotting the differential sedimentationcoefficient distribution value, C(S), versus the sedimentationcoefficient (in Svedberg units, S) yielded distinct peaks with uniquesedimentation coefficients rAd2 species (FIG. 16). The C(S) values weredetermined using the SEDFIT algorithm described by Schuck (2000)Biophys. J., 78:1606-19.

For the rAd2 vector prep shown in FIG. 16, 87.8% of the rAd2 vectorpreparation sedimented with an S value of 731, consistent with a vectorpreparation consisting predominantly of genome containing capsids. Thesedata confirm that adenoviral particles can be resolved by AUC.

What is claimed is:
 1. A method of characterizing a preparation ofrecombinant adeno-associated viral (AAV) particles comprising the stepsof a) subjecting the preparation to analytical ultracentrifugation underboundary sedimentation velocity conditions wherein the sedimentation ofrecombinant AAV particles is monitored at time intervals, b) plottingthe differential sedimentation coefficient distribution value (C(s))versus the sedimentation coefficient in Svedberg units (S), and c)integrating the area under each peak in the C(s) distribution todetermine the relative concentration of each peak, wherein each peakrepresents a species of recombinant AAV particle.
 2. A method to assessvector genome integrity of recombinant AAV particles in a preparation ofrecombinant AAV particles comprising a) subjecting the preparation toanalytical ultracentrifugation under boundary sedimentation velocityconditions wherein the sedimentation of recombinant AAV particles ismonitored at time intervals, b) plotting the differential sedimentationcoefficient distribution value C(s) versus the sedimentation coefficientin Svedberg units (S), and c) identifying species of recombinant AAVparticles in the preparation by presence of peaks on the plotcorresponding to an S value, wherein the genome size of a particularspecies of recombinant AAV particles is calculated by comparing the Svalue of the species to a standard curve generated by S values ofrecombinant AAV particles comprising encapsidated AAV genomes of knownnucleotide sizes.
 3. The method of claim 2 further comprisingintegrating the area under each peak in the C(s) distribution todetermine the relative concentration of each species of recombinant AAVparticles.
 4. A method to determine the presence of empty capsids orcapsid particles comprising variant sized recombinant AAV genomes in apreparation of recombinant AAV particles comprising the steps of a)subjecting the preparation to analytical ultracentrifugation underboundary sedimentation velocity conditions wherein the sedimentation ofrecombinant AAV particles is monitored at time intervals, and b)plotting the differential sedimentation coefficient distribution value(C(s)) versus the sedimentation coefficient in Svedberg units (S),wherein the presence of one or more peaks other than the peak for fullcapsid particles comprising intact recombinant AAV genomes indicatesthat presence of capsid particles comprising variant sized genomesand/or empty capsids.
 5. A method of measuring the relative amount emptycapsids in a preparation of recombinant AAV particles comprising thesteps of a) subjecting the preparation to analytical ultracentrifugationunder boundary sedimentation velocity conditions wherein thesedimentation of recombinant AAV particles is monitored at timeintervals, b) plotting the differential sedimentation coefficientdistribution value (C(s)) versus the sedimentation coefficient inSvedberg units (S), c) integrating the area under each peak in the C(s)distribution to determine the relative concentration of each species ofrecombinant AAV particles, and d) comparing the amount of recombinantAAV particles having an S value corresponding to empty capsid particlesto the amount of recombinant AAV particles having an S valuecorresponding to recombinant AAV particles comprising intact AAV genomesor the total amount of recombinant AAV particles in the preparation. 6.A method of measuring the relative amount of capsid particles comprisingvariant recombinant AAV genomes or empty AAV capsid particles in apreparation of recombinant AAV particles comprising the steps of a)subjecting the preparation to analytical ultracentrifugation underboundary sedimentation velocity conditions wherein the sedimentation ofrecombinant AAV particles is monitored at time intervals, b) plottingthe differential sedimentation coefficient distribution value (C(s))versus the sedimentation coefficient in Svedberg units (S), c)integrating the area under each peak in the C(s) distribution todetermine the relative concentration of each species of recombinant AAVparticles, d) comparing the amount of recombinant AAV particles havingan S values that do not correspond to recombinant AAV particlescomprising intact AAV genomes to the amount of recombinant AAV particleshaving an S value that corresponds to recombinant AAV particlescomprising intact AAV genomes or to the total amount of recombinant AAVparticles in the preparation.
 7. A method of measuring the relativeamount of capsid particles comprising variant recombinant AAV genomes ina preparation of recombinant AAV particles comprising the steps of a)subjecting the preparation to analytical ultracentrifugation underboundary sedimentation velocity conditions wherein the sedimentation ofrecombinant AAV particles is monitored at time intervals, b) plottingthe differential sedimentation coefficient distribution value (C(s))versus the sedimentation coefficient in Svedberg units (S), c)integrating the area under each peak in the C(s) distribution todetermine the relative concentration of each species of recombinant AAVparticles, d) comparing the amount of recombinant AAV particles havingan S values that do not correspond to recombinant AAV particlescomprising intact AAV genomes or empty capsid particles to the totalamount of recombinant AAV particles in the preparation.
 8. A method ofmeasuring the relative amount of recombinant AAV particles comprisingintact AAV genomes in a preparation of recombinant AAV particlescomprising the steps of a) subjecting the preparation to analyticalultracentrifugation under boundary sedimentation velocity conditionswherein the sedimentation of recombinant AAV particles is monitored attime intervals, b) plotting the differential sedimentation coefficientdistribution value (C(s)) versus the sedimentation coefficient inSvedberg units (S), c) integrating the area under each peak in the C(s)distribution to determine the relative concentration of each species ofrecombinant AAV particles, d) comparing the amount of recombinant AAVparticles having an S values corresponding to recombinant AAV particlescomprising intact AAV genomes to the amount of recombinant AAV particleshaving an S value corresponding to empty capsid particles, to capsidparticles comprising variant recombinant AAV genomes, and/or to thetotal amount of recombinant AAV particles in the preparation.
 9. Amethod of monitoring the removal of empty capsids and/or capsidparticles comprising variant recombinant AAV genomes during thepurification of a preparation of recombinant AAV particles, the methodcomprising removing a sample of the recombinant AAV particles from thepreparation following one or more steps in the purification process andanalyzing the sample for the relative amount of empty capsids and/orcapsid particles comprising variant recombinant AAV genomes according tothe method of claim 5, wherein a decrease in the relative amount ofempty capsids and/or capsids comprising variant genomes to full capsidsindicates removal of empty capsids from the preparation of recombinantAAV particles.
 10. The method of claim 4, wherein the presence of a peakthat corresponds to the S value of empty capsid particles indicates thepresence of empty capsid particles; or the presence of one or more peaksother than the peak for full capsid particles comprising intactrecombinant AAV genomes or empty capsid particles indicates thatpresence of capsid particles comprising variant sized genomes.
 11. Themethod of claim 10, wherein the presence of one or more peaks other thanthe peak for full capsid particles comprising intact recombinant AAVgenomes or empty capsid particles indicates that presence of capsidparticles comprising variant sized genomes, and wherein the capsidparticles comprising variant sized genomes comprises truncated genomes,aggregates, recombinants and/or DNA impurities compared to the intactrecombinant AAV genome.
 12. A method of determining the heterogeneity ofrecombinant AAV particles in a preparation of recombinant AAV particlescomprising the steps of a) subjecting the preparation to analyticalultracentrifugation under boundary sedimentation velocity conditionswherein the sedimentation of recombinant AAV particles is monitored attime intervals, b) plotting the differential sedimentation coefficientdistribution value (C(s)) versus the sedimentation coefficient inSvedberg units (S), wherein the presence of peaks in addition to thepeak representing capsids comprising an intact AAV genome indicatesheterogeneity of recombinant particles in the preparation.
 13. Themethod of claim 12, wherein the presence of additional peaks indicatesthe presence of empty capsid particles and/or recombinant AAV particlescomprising variant genomes.
 14. The method of claim 13, wherein thevariant genomes are truncated AAV genomes, aggregates, recombinantsand/or DNA impurities compared to the intact recombinant AAV genome. 15.The method of claim 12, further comprising integrating the area undereach peak in the C(s) distribution to determine the relativeconcentration of each species of recombinant AAV particles.
 16. A methodof monitoring the homogeneity of recombinant AAV particles during thepurification of a preparation of recombinant AAV particles, the methodcomprising removing a sample of the recombinant AAV particles from thepreparation following one or more steps in the purification process anddetermining the heterogeneity of recombinant AAV particles according tothe method of claim 15, wherein an increase in the relative amount ofrecombinant AAV particles comprising intact viral genomes indicates anincrease in the homogeneity of full AAV particles in the preparation ofrecombinant AAV particles.
 17. The method of claim 1, wherein thesedimentation of recombinant AAV particles is monitored by absorbance orinterference.
 18. The method of claim 1, wherein the preparation is anaqueous solution.
 19. The method of claim 18, wherein the monitoringfurther comprises comparison to a reference sample, wherein thereference sample comprises the aqueous solution without recombinant AAVparticles.
 20. The method of claim 1, wherein the C(s) values aredetermined by an algorithm that comprises Lamm equation solutions. 21.The method of claim 20, wherein the algorithm is the SEDFIT algorithm.22. The method of claim 1, wherein: sedimentation is monitored until therecombinant AAV particles with the lowest density sediments to thebottom of a sector of an ultracentrifuge.
 23. The method of claim 1,wherein the ultracentrifugation utilizes an ultracentrifuge comprisingan ultracentrifuge velocity cell.
 24. The method of claim 1, whereinsedimentation is monitored until recombinant AAV particles sediment tothe bottom of an ultracentrifuge velocity cell.
 25. The method of claim23, wherein sedimentation is monitored until the recombinant AAVparticles with the lowest density sediments and clears an opticalwindow.
 26. The method of claim 22, wherein at least 30 scans are usedto monitor sedimentation of recombinant AAV particles.
 27. The method ofclaim 20, wherein a regularization is applied to a fitting level with aconfidence level of F statistic of at least about 0.68.
 28. The methodof claim 27, wherein the regularization is a second derivativeregularization.
 29. The method of claim 27, wherein the regularizationis Max entropy regularization.
 30. The method of claim 20, wherein thefollowing C(S) parameters are held constant: resolution of about 200 Sto about 5000 S, S min is about 1 S to about 100 S, S max is about 100 Sto about 5000 S, and frictional ratio is about 1.0 or is left to floatto a value determined by centrifugation software.
 31. The method ofclaim 1, wherein the sedimentation of recombinant AAV particles ismonitored about every 10-60 seconds.
 32. The method of claim 1, whereinthe boundary sedimentation velocity is performed at about 3,000 rpm toabout 20,000 rpm and/or at about 4° C. to about 20° C.
 33. A method ofevaluating a process for the production of recombinant AAV particlescomprising the method of claim 1, wherein an increase in the relativeamount of recombinant AAV particles comprising intact AAV genomescompared to the relative amount of empty capsid particles and/orrecombinant AAV capsid particles with variant recombinant AAV genomescompared to a reference preparation of recombinant AAV particlesindicates an improvement in the production of recombinant AAV particles.34. A method for preparing recombinant AAV particles with reduced emptycapsids and/or recombinant AAV particles comprising variant genomes, themethod comprising a) culturing host cells under conditions suitable forrecombinant AAV production, wherein the cells comprise i) nucleic acidencoding a heterologous transgene flanked by at least one AAV ITR, ii)nucleic acid comprising AAV rep and cap coding regions, wherein thenucleic acid comprises a mutated p5 promoter wherein rep expression fromthe p5 promoter is reduced compared to a wild-type p5 promoter, and iii)nucleic acid encoding AAV helper virus functions; b) lysing the hostcells to release recombinant AAV particles; c) isolating the recombinantAAV particles produced by the host cell; and d) analyzing therecombinant AAV particles for the presence of empty capsids and/orrecombinant AAV particles with variant genomes by analyticalultracentrifugation by the method of claim 1.